Seasonal impact of PM(2.5) on pro-oncogenic drivers among participants of a thermal power plant in India.

SUBJECT:ghosh-powerplant-participants

Asymptomatic male thermal power plant employees, Bankura district, West Bengal, India

352 individuals screened, 88 asymptomatic, 33 adult males consented and were sampled in both seasons. Inclusion: asymptomatic males aged 25-60 years with no tobacco, alcohol, gutka or pan habit, working at the plant 8-10 h/day for at least 5 years. Exclusion: any diagnosed chronic disease, current medication, history of malignancy, oral contraceptives, pregnancy or lactation. The cohort was 100% male (female participation was inadequate), mean BMI 24.91 +/- 3.92 kg/m2, mean 13.38 +/- 6.09 years of service, mean 8.87 +/- 1.86 h work per day; 75.75% office employees and 24.24% plant employees; 81.33% reported no passive smoke exposure and the 18.67% who did were carried as an LMER covariate without changing the PM2.5 associations.

Paired within-subject panel study: the same participants were examined once in Aug 2021 (monsoon, after the low-PM2.5 summer-monsoon months) and once in Mar 2022 (spring, after the high-PM2.5 winter-spring months). Study approved by the Institutional Ethics Committee (CNCI-IEC-DS-2019-10) and conducted per the Declaration of Helsinki.

SUBJECT:ghosh-powerplant-sputum

Spontaneous sputum from asymptomatic male thermal power plant employees

Same 33 participants sampled in both seasons; sputum-derived cells are referred to as airway cells throughout the paper.

Paired monsoon (Aug 2021) and spring (Mar 2022) sampling

SUBJECT:ghosh-powerplant-leukocytes

Whole-blood leukocytes from asymptomatic male thermal power plant employees

Same 33 participants sampled in both seasons; the leukocytes represent the systemic cellular environment.

SUBJECT:ghosh-powerplant-serum

Serum from asymptomatic male thermal power plant employees

Same 33 participants sampled in both seasons.

LF:ghosh-pm25-seasonal-spirometry

Spirometry in thermal power plant participants during monsoon and spring

Measured and percent-predicted FVC, FEV1 and FEV1/FVC in the same 33 asymptomatic male participants at the low-PM2.5 monsoon visit (Aug 2021) and the high-PM2.5 spring visit (Mar 2022). FVC and FEV1 stayed within normal limits and did not differ significantly between seasons (p = 0.59 and p = 0.66), but the measured FEV1/FVC ratio fell significantly in spring (90.06 +/- 6.19% to 84.32 +/- 3.86%, p = 0.00), which the authors read as a tendency toward obstructive lung function. Percent-predicted values, reported in Table 3 but not recorded in the output slots because the schema has no percent-predicted slot, were: FVC 88.67 +/- 7.4% (monsoon) and 87.06 +/- 14.06% (spring); FEV1 100.15 +/- 6.39% and 100.55 +/- 15.61%; FEV1/FVC 109.85 +/- 9.28% and 109.48 +/- 10.66%. HEMATOLOGY CONTEXT (Table 2, same visits, N = 33 each, paired t-test): Hb 13.68 +/- 1.36 to 12.95 +/- 1.28 g/dl (p = 0.00); ESR 28.61 +/- 25.65 to 17.24 +/- 18.83 mm (p = 0.00); WBC 7603.03 +/- 1628.55 to 5551.52 +/- 2383.42 per cu. mm (p = 0.00); monocytes 1.03 +/- 0.18% to 2.19 +/- 1.6% (p = 0.00); eosinophils, lymphocytes and neutrophils unchanged. The corresponding LMER estimates per 10 ug/m3 PM2.5 were Hb -0.33 (95% CI -0.45, -0.21), ESR -5.19 (95% CI -8.46, -1.92) and WBC -937.49 (95% CI -1351.36, -523.62). UNITS: Table 3 prints bare numbers with no unit column, so the litre unit on FVC and FEV1 and the percent unit on FEV1/FVC are the conventional spirometry units rather than units the paper states. These hematology values are recorded here as description text only: the schema has no whole-blood hematology output class, and LungFunctionAssay's hemoglobin_level slot sits on the assay rather than on the per-season output records, so it cannot carry two seasonal values.

Exposure conditions

Outputs

Evidence

BALF:ghosh-sputum-cytology

Sputum differential cytology during monsoon and spring

Papanicolaou-stained spontaneous sputum from the same 33 participants in both seasons, counted over at least 10 random fields per sample. Alveolar macrophages - the primary cells recruited against respiratory pathogens - were significantly reduced in spring, which the authors read as reduced pulmonary immunity. Neutrophils trended upward in spring but the seasonal difference was not significant. The LMER model estimated a decrease in the frequency of alveolar macrophages of -5.46 (95% CI -9.44, -1.47) per 10 ug/m3 rise in PM2.5, adjusted for the independent covariates. Fig. 2a, b reports the average cell numbers graphically only, with no numeric values in the text; the paper's unit is an average cell number per field rather than a percentage, so no value is recorded in the percentage slots; the LMER estimate is recorded as COMP:ghosh-sputum-alveolar-macrophages with its unit omitted and its scale explained.

Exposure conditions

Outputs

Evidence

OX:ghosh-ros-airway

Intracellular ROS in sputum-derived airway cells during monsoon and spring

DCFH-DA mean fluorescence intensity in airway cells (N = 33), normalized per 10^5 viable cells. Spring samples recorded a greater amount of ROS production than monsoon samples. The LMER model estimated a rise of 0.064 (95% CI 0.045, 0.078) in airway-cell ROS per 10 ug/m3 increase in PM2.5. MFI values are shown in Fig. 2e only, so no value is recorded in the output records. Pearson correlation between MMP-9 activity and ROS was not significant in either compartment, which the authors attribute to ROS being one of several non-linear influences on MMP-9.

Exposure conditions

Outputs

Evidence

OX:ghosh-ros-leukocyte

Intracellular ROS in whole-blood leukocytes during monsoon and spring

DCFH-DA mean fluorescence intensity in leukocytes (N = 33), normalized per 10^5 viable cells. Spring samples recorded a greater amount of ROS production than monsoon samples. The LMER model estimated a rise of 0.046 (95% CI 0.034, 0.056) in leukocyte ROS per 10 ug/m3 increase in PM2.5. MFI values are shown in Fig. 2f only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-pik3r1

PIK3R1 (PI3K p85alpha) phosphorylation in sputum-derived airway cells during monsoon and spring

Phosphorylated PI3K p85alpha Tyr508 in sputum-derived airway cells, read by indirect ELISA and normalized to its total protein counterpart. PI3K activity was significantly increased in spring, evident from increased pPI3K p85alpha Tyr508 phosphorylation. LMER estimated +1.91 (95% CI 1.43, 2.39) relative units per 10 ug/m3 increase in PM2.5. Values are in Fig. 3c only, so no value is recorded in the output records. The readout is the p85alpha regulatory subunit - the PIK3R1 gene product - so this assay is tagged PIK3R1 (NCBIGene:5295) and not the p110alpha catalytic subunit PIK3CA; no PIK3CA transcript was measured in the airway cells (it was measured in the leukocytes, see GE:ghosh-leukocyte-pik3ca).

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-akt2

AKT2 expression in sputum-derived airway cells during monsoon and spring

AKT2 transcript (delta-Ct, GAPDH reference) and pAKT Ser473 protein in airway cells. AKT2 mRNA expression was increased in spring versus monsoon, and AKT1/2/3 activity rose as shown by increased pAKT Ser473 phosphorylation. LMER estimated +1.76 (95% CI 1.46, 2.05) relative units of airway pAKT Ser473 per 10 ug/m3 increase in PM2.5. No LMER estimate is reported for airway AKT2 mRNA; both readouts appear in Figs. 3a and 3c only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-mtor

MTOR expression in sputum-derived airway cells during monsoon and spring

MTOR transcript in airway cells (delta-Ct, GAPDH reference). MTOR mRNA expression was increased in spring versus monsoon. After Bonferroni adjustment the airway MTOR delta-Ct differed significantly between the two PM2.5 exposure levels, and LMER estimated a delta-Ct change of -1.35 (95% CI -1.84, -0.88) per 10 ug/m3 increase in PM2.5 - a negative delta-Ct shift, meaning higher transcript abundance. Values are in Fig. 3a only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-pten

PTEN expression in sputum-derived airway cells during monsoon and spring

PTEN - the tumour suppressor and negative regulator of PI3K - in airway cells. Transcript expression decreased and protein expression decreased in spring versus monsoon. After Bonferroni adjustment the airway PTEN delta-Ct differed significantly between the two PM2.5 exposure levels, and LMER estimated a delta-Ct change of +5.36 (95% CI 4.95, 5.77) per 10 ug/m3 increase in PM2.5 - a positive delta-Ct shift, meaning lower transcript abundance. No LMER estimate is reported for airway PTEN protein. Values are in Figs. 3a and 3d only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-egfr

EGFR expression in sputum-derived airway cells during monsoon and spring

EGFR transcript in airway cells (delta-Ct, GAPDH reference). EGFR mRNA did not change significantly between monsoon and spring in the airway cells, even though EGFR protein in the same cells was upregulated during spring (recorded separately in EGFR:ghosh-airway-egfr-signaling). No LMER estimate is reported for airway EGFR mRNA. Values are in Fig. 4c only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-bcl2

BCL2 expression in sputum-derived airway cells during monsoon and spring

The anti-apoptotic gene BCL2 in airway cells. BCL2 transcript was significantly upregulated in spring and Bcl-2 protein showed robust expression in spring, so Bcl-2 upregulation was consistent at both transcript and protein level in the airway cells. After Bonferroni adjustment the airway BCL2 delta-Ct differed significantly between the two PM2.5 exposure levels; LMER estimated a delta-Ct change of -1.61 (95% CI -2.14, -1.09) - higher transcript abundance - and a protein change of +0.82 (95% CI 0.69, 0.94) relative units per 10 ug/m3 increase in PM2.5. Airway cells also showed a significant decline in the frequency of early apoptotic cells in spring, with an LMER estimate of -11.00 (95% CI -14.27, -7.77) per 10 ug/m3 PM2.5; that flow-cytometry endpoint is also recorded as COMP:ghosh-airway-early-apoptotic-cells. Values are in Figs. 3g, 3i and 3k only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-bax

BAX expression in sputum-derived airway cells during monsoon and spring

The pro-apoptotic gene BAX in airway cells. BAX transcript remained unaltered in the airway cells between seasons, while BAX protein showed robust spring expression - a transcript/protein discordance the authors attribute to post-transcriptional regulation, enhanced protein stability or reduced proteasomal degradation. LMER estimated a BAX protein change of +1.66 (95% CI 1.49, 1.82) relative units per 10 ug/m3 increase in PM2.5; no LMER estimate is reported for airway BAX mRNA. Values are in Figs. 3i and 3k only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-ccnd1

CCND1 expression in sputum-derived airway cells during monsoon and spring

The proliferation marker Cyclin D1 (CCND1) in airway cells. CCND1 mRNA showed no significant seasonal difference, but Cyclin D1 protein was prominently expressed in spring, which the authors read as increased proliferative action. LMER estimated a Cyclin D1 protein change of +2.87 (95% CI 2.26, 3.6) relative units per 10 ug/m3 increase in PM2.5; no LMER estimate is reported for airway CCND1 mRNA. Values are in Figs. 3i and 3k only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-vegfa

VEGFA expression in sputum-derived airway cells during monsoon and spring

The pro-angiogenic downstream target VEGFA in airway cells. VEGFA showed lower delta-Ct values in spring - that is, relatively higher transcript abundance - and VEGF protein was validated as upregulated in the airway cells during spring. After Bonferroni adjustment the airway VEGFA delta-Ct differed significantly between the two PM2.5 exposure levels; LMER estimated a delta-Ct change of -2.11 (95% CI -2.67, -1.55) and a protein change of +1.60 (95% CI 1.23, 1.97) relative units per 10 ug/m3 increase in PM2.5. Values are in Figs. 3m and 3o only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-airway-hif1a

HIF1A expression in sputum-derived airway cells during monsoon and spring

The pro-angiogenic downstream target HIF1A in airway cells. HIF1A showed lower delta-Ct values in spring - relatively higher transcript abundance - and HIF-1alpha protein was validated as upregulated in the airway cells during spring. After Bonferroni adjustment the airway HIF1A delta-Ct differed significantly between the two PM2.5 exposure levels; LMER estimated a delta-Ct change of -2.02 (95% CI -3.15, -0.87) and a protein change of +0.02 (95% CI 0.01, 0.03) relative units per 10 ug/m3 increase in PM2.5. Values are in Figs. 3m and 3o only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-pik3ca

PIK3CA expression in whole-blood leukocytes during monsoon and spring

PIK3CA transcript and phosphorylated PI3K p85alpha Tyr508 protein in whole-blood leukocytes. Leukocytes showed higher PIK3CA transcript abundance in spring, and increased pPI3K p85alpha Tyr508 phosphorylation was also observed. LMER estimated a PIK3CA delta-Ct change of -1.01 (95% CI -1.45, -0.56) - higher transcript abundance - and a pPI3K p85alpha Tyr508 protein change of +1.62 (95% CI 1.22, 2.02) relative units per 10 ug/m3 increase in PM2.5. The transcript readout is PIK3CA (the p110alpha catalytic subunit) while the protein readout is p85alpha (the PIK3R1 gene product); the paper reports the two PI3K readouts together, and this assay is tagged PIK3CA for the transcript it actually measures. Values are in Figs. 3b and 3e only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-akt2

AKT2 expression in whole-blood leukocytes during monsoon and spring

Phosphorylated AKT Ser473 in whole-blood leukocytes, read by indirect ELISA and normalized to its total protein counterpart. Increased pAKT Ser473 phosphorylation was observed in the leukocytes during spring. LMER estimated +1.34 (95% CI 0.93, 1.76) relative units per 10 ug/m3 increase in PM2.5. AKT2 transcript was measured in the airway cells rather than the leukocytes. Values are in Fig. 3e only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-mtor

MTOR expression in whole-blood leukocytes during monsoon and spring

MTOR transcript in whole-blood leukocytes (delta-Ct, GAPDH reference). Leukocytes showed higher MTOR transcript abundance in spring than monsoon. No LMER estimate is reported for leukocyte MTOR. Values are in Fig. 3b only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-pten

PTEN expression in whole-blood leukocytes during monsoon and spring

PTEN in whole-blood leukocytes. Leukocytes showed lower PTEN transcript abundance in spring and PTEN protein showed decreased expression. LMER estimated a PTEN delta-Ct change of +4.52 (95% CI 4.01, 5.03) - lower transcript abundance - and a PTEN protein change of -0.12 (95% CI -0.24, 0.01) relative units per 10 ug/m3 increase in PM2.5. Values are in Figs. 3b and 3f only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-egfr

EGFR expression in whole-blood leukocytes during monsoon and spring

EGFR transcript in whole-blood leukocytes (delta-Ct, GAPDH reference). Robust EGFR expression at transcript level was observed in the leukocytes during spring. LMER confirmed that a rise in PM2.5 of 10 ug/m3 increased leukocyte EGFR mRNA levels, with a delta-Ct estimate of -1.76 (95% CI -2.28, -1.25) - a negative delta-Ct shift, meaning higher transcript abundance. Values are in Fig. 4e only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-bcl2

BCL2 expression in whole-blood leukocytes during monsoon and spring

The anti-apoptotic gene BCL2 in whole-blood leukocytes. BCL2 transcript remained unaltered between seasons while Bcl-2 protein showed robust spring expression. LMER estimated a Bcl-2 protein change of +0.67 (95% CI 0.47, 0.88) relative units per 10 ug/m3 increase in PM2.5; no LMER estimate is reported for leukocyte BCL2 mRNA. The leukocytes also showed a significant decrease in both early and late apoptosis during spring; that flow-cytometry endpoint has no matching output slot, so it is recorded here as description text. Values are in Figs. 3h, 3j and 3l only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-bax

BAX expression in whole-blood leukocytes during monsoon and spring

The pro-apoptotic gene BAX in whole-blood leukocytes. BAX transcript showed significantly low levels in spring, evident from increased delta-Ct values, while BAX protein showed robust spring expression. LMER estimated a BAX delta-Ct change of +1.24 (95% CI 0.64, 1.83) - lower transcript abundance - and a BAX protein change of +0.57 (95% CI 0.41, 0.73) relative units per 10 ug/m3 increase in PM2.5. Values are in Figs. 3j and 3l only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-ccnd1

CCND1 expression in whole-blood leukocytes during monsoon and spring

The proliferation marker Cyclin D1 (CCND1) in whole-blood leukocytes. CCND1 mRNA showed no significant seasonal difference, but Cyclin D1 protein was highly upregulated in spring. LMER estimated a Cyclin D1 protein change of +1.29 (95% CI 1.05, 1.54) relative units per 10 ug/m3 increase in PM2.5; no LMER estimate is reported for leukocyte CCND1 mRNA. Values are in Figs. 3j and 3l only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-vegfa

VEGFA expression in whole-blood leukocytes during monsoon and spring

The pro-angiogenic downstream target VEGFA in whole-blood leukocytes. VEGFA showed lower delta-Ct values in spring - relatively higher transcript abundance - and VEGF protein was validated as upregulated in the leukocytes during spring. LMER estimated a VEGF protein change of +1.08 (95% CI 0.71, 1.45) relative units per 10 ug/m3 increase in PM2.5; no LMER estimate is reported for leukocyte VEGFA mRNA. Values are in Figs. 3n and 3p only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-leukocyte-hif1a

HIF1A expression in whole-blood leukocytes during monsoon and spring

The pro-angiogenic downstream target HIF1A in whole-blood leukocytes. HIF1A showed lower delta-Ct values in spring - relatively higher transcript abundance - and HIF-1alpha protein was validated as upregulated in the leukocytes during spring. LMER estimated a HIF1A delta-Ct change of -0.99 (95% CI -1.60, -0.36) and a HIF-1alpha protein change of +0.05 (95% CI 0.04, 0.06) relative units per 10 ug/m3 increase in PM2.5. Values are in Figs. 3n and 3p only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

GE:ghosh-serum-mmp9-pro

MMP9-pro expression in serum during monsoon and spring

Pro MMP-9 (latent form) activity in serum, measured by gelatin zymography and quantified by ImageJ band densitometry. Pro MMP-9 activity decreased from monsoon to spring. LMER estimated a change of -19.42 (95% CI -22.68, -16.16) band-intensity units per 10 ug/m3 increase in PM2.5. Values are in Figs. 2c and 2d only, so no value is recorded in the output records. Pearson correlation between MMP-9 activity and ROS generation was not significant in either airway cells or leukocytes.

Exposure conditions

Outputs

Evidence

GE:ghosh-serum-mmp9-active

MMP9-active expression in serum during monsoon and spring

Active MMP-9 activity in serum, measured by gelatin zymography and quantified by ImageJ band densitometry. Active MMP-9 activity increased significantly from monsoon to spring. LMER estimated a change of +26.89 (95% CI 5.89, 47.90) band-intensity units per 10 ug/m3 increase in PM2.5. Values are in Figs. 2c and 2d only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

EGFR:ghosh-airway-egfr-signaling

EGFR phosphorylation, total protein and localization in sputum-derived airway cells

EGFR in airway cells across the paired seasonal visits. Immunocytochemistry showed increased subcellular EGFR localization in the sputum-derived cells during spring. EGFR mRNA in the airway cells did not change significantly between seasons, but EGFR protein in the same cells was upregulated during spring. LMER estimated, per 10 ug/m3 rise in PM2.5, an increase of 2.31 (95% CI 2.06, 2.56) in airway pEGFR Tyr1068 by ELISA and of 13.90 (95% CI 2.29, 25.5) in EGFR localization by immunocytochemistry. Recruitment of pEGFR Tyr1068 is the authors' chosen marker because it is well known for EGFR sensitizing mutations in lung cancer. All values appear in Fig. 4a-d only, so no value is recorded in the output records.

Exposure conditions

Outputs

Evidence

EGFR:ghosh-leukocyte-egfr-signaling

EGFR phosphorylation and total protein in whole-blood leukocytes

EGFR in leukocytes across the paired seasonal visits. Robust EGFR expression was seen at both transcript and protein level in the leukocytes during spring. LMER estimated an increase of 1.42 (95% CI 1.14, 1.71) in leukocyte pEGFR Tyr1068 by ELISA per 10 ug/m3 rise in PM2.5. Values appear in Fig. 4e-f only, so no value is recorded in the output records; the transcript result is recorded separately in GE:ghosh-leukocyte-egfr.

Exposure conditions

Outputs

Evidence

COMP:ghosh-lf-fev1fvc

Association of PM2.5 with the measured FEV1/FVC ratio

LMER beta estimate: every 10 ug/m3 increase in PM2.5 decreased the measured FEV1/FVC ratio by 2.62 percentage points.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-ros-airway

Association of PM2.5 with ROS generation in airway cells

LMER beta estimate: every 10 ug/m3 rise in PM2.5 caused robust ROS generation in the airway cells. Unit omitted: the readout is DCFH-DA mean fluorescence intensity normalized per 10^5 viable cells, for which no unit ontology term applies.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-ros-leukocyte

Association of PM2.5 with ROS generation in leukocytes

LMER beta estimate: every 10 ug/m3 rise in PM2.5 caused robust ROS generation in the leukocytes. Unit omitted: the readout is DCFH-DA mean fluorescence intensity normalized per 10^5 viable cells.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-egfr-airway-pegfr-y1068

Association of PM2.5 with pEGFR Tyr1068 in airway cells

LMER beta estimate for airway-cell pEGFR Tyr1068 by ELISA. Unit omitted: ELISA readouts are normalized relative units, not absolute concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-egfr-airway-localization

Association of PM2.5 with EGFR localization in sputum-derived cells

LMER beta estimate for EGFR localization as measured by immunocytochemistry mean fluorescence intensity. Unit omitted: the readout is an ImageJ region-of-interest MFI.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-egfr-leukocyte-pegfr-y1068

Association of PM2.5 with pEGFR Tyr1068 in leukocytes

LMER beta estimate for leukocyte pEGFR Tyr1068 by ELISA. Unit omitted: ELISA readouts are normalized relative units.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-mtor-mrna

Association of PM2.5 with MTOR transcript (delta-Ct) in airway cells

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-pten-mrna

Association of PM2.5 with PTEN transcript (delta-Ct) in airway cells

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-bcl2-mrna

Association of PM2.5 with BCL2 transcript (delta-Ct) in airway cells

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-hif1a-mrna

Association of PM2.5 with HIF1A transcript (delta-Ct) in airway cells

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-vegfa-mrna

Association of PM2.5 with VEGFA transcript (delta-Ct) in airway cells

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-pik3r1-protein

Association of PM2.5 with PIK3R1 (pPI3K p85alpha Tyr508) protein in airway cells

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-akt2-protein

Association of PM2.5 with pAKT Ser473 protein in airway cells

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-ccnd1-protein

Association of PM2.5 with Cyclin D1 protein in airway cells

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-bcl2-protein

Association of PM2.5 with Bcl-2 protein in airway cells

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-bax-protein

Association of PM2.5 with BAX protein in airway cells

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-hif1a-protein

Association of PM2.5 with HIF-1alpha protein in airway cells

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-vegfa-protein

Association of PM2.5 with VEGF protein in airway cells

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-pik3ca-mrna

Association of PM2.5 with PIK3CA transcript (delta-Ct) in leukocytes

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-hif1a-mrna

Association of PM2.5 with HIF1A transcript (delta-Ct) in leukocytes

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-pten-mrna

Association of PM2.5 with PTEN transcript (delta-Ct) in leukocytes

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-bax-mrna

Association of PM2.5 with BAX transcript (delta-Ct) in leukocytes

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-egfr-mrna

Association of PM2.5 with EGFR transcript (delta-Ct) in leukocytes

change_direction refers to mrna_level - transcript abundance - because that is what compared_measurement names. change_value is the LMER beta on the delta-Ct scale (Ct_target - Ct_GAPDH), which runs INVERSELY to transcript abundance, so its sign is deliberately opposite to change_direction: a negative beta means delta-Ct fell, i.e. transcript abundance ROSE with PM2.5, and a positive beta means transcript abundance FELL. Unit omitted: delta-Ct is a PCR cycle difference with no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-pik3ca-protein

Association of PM2.5 with pPI3K p85alpha Tyr508 protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-akt2-protein

Association of PM2.5 with pAKT Ser473 protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-ccnd1-protein

Association of PM2.5 with Cyclin D1 protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-bcl2-protein

Association of PM2.5 with Bcl-2 protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-bax-protein

Association of PM2.5 with BAX protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-hif1a-protein

Association of PM2.5 with HIF-1alpha protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-vegfa-protein

Association of PM2.5 with VEGF protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-leukocyte-pten-protein

Association of PM2.5 with PTEN protein in leukocytes

LMER beta estimate on the normalized relative-unit scale of the indirect ELISA readout. Unit omitted: the ELISA reports normalized relative units, not absolute protein concentrations. NOTE: the 95% CI (-0.24, 0.01) spans zero, so this association is not statistically significant even though the paper describes PM2.5 as having 'decreased the leukocyte protein PTEN'; change_direction follows the paper’s wording.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-serum-mmp9-pro-protein

Association of PM2.5 with pro MMP-9 protein in serum

LMER beta estimate on the ImageJ band-intensity scale of the gelatin zymogram. Unit omitted: band intensity has no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-serum-mmp9-active-protein

Association of PM2.5 with active MMP-9 protein in serum

LMER beta estimate on the ImageJ band-intensity scale of the gelatin zymogram. Unit omitted: band intensity has no unit ontology term.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-sputum-alveolar-macrophages

Association of PM2.5 with the frequency of alveolar macrophages in sputum

LMER beta estimate for the frequency of alveolar macrophages in spontaneous sputum. Unit omitted and the slot name overstates the scale: the paper reports an average cell number per counted field (Fig. 2b legend), not a percentage, and macrophage_percentage is the only macrophage slot BALFSputumOutput offers. Recorded here rather than left in prose so the number is queryable alongside the other LMER estimates.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

COMP:ghosh-airway-early-apoptotic-cells

Association of PM2.5 with the frequency of early apoptotic cells in airway cells

LMER beta estimate for the decrement of early apoptotic airway cells by annexin V-FITC/PI flow cytometry. Attached to GE:ghosh-airway-bcl2 because that assay carries the apoptosis readouts for this compartment; percentage_positive_cells is the nearest GeneExpressionOutput slot. Unit omitted: the paper reports an average number of early apoptotic cells (Fig. 3g legend) rather than a percentage. Recorded here rather than left in prose so the number is queryable.

Statistical test: linear mixed-effects regression (LMER) with participant identity code as the random effect, adjusted for age, BMI, marital status, number of family members, education, profession, income, number of working years, work duration, mode of transport, passive smoking and cooking fuel type; the estimate is the change per 10 ug/m3 increase in PM2.5, not a raw monsoon-to-spring difference

KER:ghosh-pi3k-akt-to-suppressed-apoptosis

PI3K/AKT pathway activation leads to suppressed apoptosis

KER:ghosh-pi3k-akt-to-proliferation

PI3K/AKT pathway activation leads to increased cell proliferation

KER:ghosh-pi3k-akt-to-proangiogenic-signaling

PI3K/AKT pathway activation leads to increased pro-angiogenic signaling

KER:ghosh-egfr-to-pi3k-akt-activation

EGFR activation leads to PI3K/AKT pathway activation

PROTOCOL:ghosh-spirometry-001

PC spirometry per American Thoracic Society guidelines

Pulmonary function testing performed according to the American Thoracic Society (1995) guidelines by PC spirometry (SP260, Schiller, Barr, Switzerland). Measured and percent-predicted FVC, FEV1 and FEV1/FVC recorded for each participant in both seasons (N = 33). PFT camps were organised on site and the reports were read by a consultant pulmonologist.

Equipment: PC spirometer (SP260, Schiller, Barr, Switzerland)

PROTOCOL:ghosh-hematology-001

Automated hematology analyzer for total count, differential count, ESR and hemoglobin

Hematological parameters - total count (TC), differential count (DC), erythrocyte sedimentation rate (ESR) and hemoglobin (Hb) - measured on an automated hematology analyzer (Erma PCE-210, Saitama, Japan) in anticoagulated whole blood collected in K2EDTA vacutainers.

Equipment: Automated hematology analyzer (Erma PCE-210, Saitama, Japan)

PROTOCOL:ghosh-pap-cytology-001

Papanicolaou staining and differential cytology of spontaneous sputum

Sputum cytology by the Papanicolaou (PAP) staining method. The average number of cells (excluding squamous epithelial cells) was calculated after counting at least 10 randomly selected fields under a light microscope (CX40, Olympus/Evident, Tokyo, Japan). Samples were accepted as representative of lower airway origin when squamous epithelial cell contamination was below 20% and bronchial epithelial cells and/or alveolar macrophages were present. Alveolar macrophages, bronchial epithelial cells, neutrophils, eosinophils and lymphocytes were identified by standard cytological criteria.

Equipment: Light microscope (CX40, Olympus/Evident, Tokyo, Japan)

PROTOCOL:ghosh-cell-isolation-001

Isolation of sputum-derived airway cells and whole-blood leukocytes

Sputum treated with dithiothreitol and centrifuged at 1000 x g for 5-6 min at room temperature; the viscous fraction diluted in PBS and centrifuged at 3000 x g for 5-6 min to pellet airway cells, then passed through 40-um cell strainers. RNA and protein extracted with the bashing-bead method (ZR BashingBead Lysis Tubes, 0.1 and 0.5 mm, Zymo Research). Anticoagulated whole blood incubated with RBC lysis buffer for 15 min and centrifuged at 3000 x g for 5 min; the leukocyte pellet washed in PBS and re-centrifuged. Lysates prepared in RIPA buffer with phosphatase inhibitors (NaF, Na3VO4) and cOmplete Mini EDTA-free protease inhibitor cocktail (Roche). Total protein determined by Lowry's method.

Equipment: Refrigerated centrifuge; 40-um cell strainers; ZR BashingBead Lysis Tubes (Zymo Research)

PROTOCOL:ghosh-zymography-mmp9-001

Gelatin zymography for pro and active MMP-9 in serum

Serum incubated with Sepharose beads (GE Healthcare 17-0956-11) for 2-3 h and run on SDS-PAGE gels containing 0.1% gelatin (Merck Sigma Aldrich K21568978). Gels washed with 2.5% Triton X-100 (Promega H5141) for 30 min and incubated in reaction buffer A (50 mM Tris-HCl pH 8.0, 0.2 M NaCl, 5 mM CaCl2) at 37 C for 24 h, then stained with Coomassie Brilliant Blue R-250 (Merck Sigma Aldrich 42660). Bands imaged on an iBright FL1500 system and quantified with ImageJ Launcher 1.4.3.67. Serum-free medium of MDA-MB-231 served as the positive control and reaction buffer B (with 20 mM EDTA) as the negative control.

Equipment: SDS-PAGE apparatus; Gel documentation system (iBright FL1500, Thermo Fisher Scientific); ImageJ Launcher version 1.4.3.67

PROTOCOL:ghosh-dcfhda-ros-001

DCFH-DA fluorescence spectrometry for intracellular ROS

Intracellular ROS measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). A 10 mM stock in anhydrous DMSO was diluted in HEPES buffer (SRL 63732) to 10 uM DCFH-DA containing 0.1% DMSO, with a DMSO vehicle control in every assay. Cell suspensions were incubated with the diluted reagent at 37 C for 45 min and read on a Cary Eclipse Fluorescence Spectrometer (Agilent). ROS reported as mean fluorescence intensity (MFI) averaged across airway cells (N = 33) and leukocytes (N = 33), normalized to viable cell count per 10^5 cells by trypan blue exclusion, with unstained-control autofluorescence subtracted.

Equipment: Cary Eclipse Fluorescence Spectrometer (Agilent, Santa Clara, CA, USA)

PROTOCOL:ghosh-annexinv-apoptosis-001

Annexin V-FITC / propidium iodide flow cytometry for apoptosis

Airway cells and leukocytes assessed with the BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (556547). Cells washed in ice-cold PBS, suspended in binding buffer, and incubated with annexin V-FITC and propidium iodide for 15 min at room temperature in the dark. Acquired on a DxFLEX flow cytometer (Beckman Coulter) with CytExpert software, 10,000 events per sample. Unstained-control autofluorescence subtracted; single-colour FITC-only and PI-only controls used for compensation and gating.

Equipment: DxFLEX flow cytometer (Beckman Coulter) with CytExpert software; BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (556547)

PROTOCOL:ghosh-icc-egfr-001

Immunocytochemistry for EGFR localization in sputum-derived airway cells

Air-dried sputum smears fixed in 95% ethanol, washed in PBS, blocked with 1% BSA in PBST and incubated overnight at 4 C with anti-human EGFR mouse monoclonal antibody (Santa Cruz sc-373746, 1:1000 in 1% BSA/PBS), then with FITC-tagged secondary antibody (Santa Cruz sc-65218, 1:2000) and DAPI (Merck Sigma Aldrich D9542). Viewed on a fluorescence microscope (MVX10, Olympus, Tokyo, Japan). No-primary-antibody controls ruled out nonspecific and autofluorescent signal. Mean fluorescence intensity quantified with ImageJ Launcher 1.4.3.67 by region-of-interest analysis across five randomly selected fields per sample.

Equipment: Fluorescence microscope (MVX10, Olympus, Tokyo, Japan); ImageJ Launcher version 1.4.3.67

PROTOCOL:ghosh-qpcr-pik3ca

RT-qPCR for PIK3CA (delta-Ct against GAPDH)

Total RNA from airway cells and leukocytes isolated with the PureLink RNA Mini Kit (12183018A) and reverse transcribed with the RevertAid RT kit (K1622); only RNA with A260/A280 between 1.8 and 2.0 was used, 2 ug reverse transcribed per reaction. PIK3CA-specific primers (Suppl. Table 4) with FastStart Essential DNA Green Master Mix (06402712001) on a LightCycler 96 (Roche). Each 15 uL reaction held 10 ng cDNA: 98 C for 10 min, then 40 cycles of 95 C for 30 s, target-specific annealing for 40 s and 72 C for 60 s, followed by melt-curve analysis. Triplicate reactions, with no-template and no-RT controls. Relative expression reported as delta-Ct (Ct_target - Ct_GAPDH); lower delta-Ct means higher transcript abundance. Results are relative, semi-quantitative trends, not fold changes.

PROTOCOL:ghosh-qpcr-akt2

RT-qPCR for AKT2 (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the AKT2-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-mtor

RT-qPCR for MTOR (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the MTOR-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-pten

RT-qPCR for PTEN (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the PTEN-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-egfr

RT-qPCR for EGFR (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the EGFR-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-bcl2

RT-qPCR for BCL2 (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the BCL2-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-bax

RT-qPCR for BAX (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the BAX-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-ccnd1

RT-qPCR for CCND1 (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the CCND1-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-vegfa

RT-qPCR for VEGFA (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the VEGFA-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-qpcr-hif1a

RT-qPCR for HIF1A (delta-Ct against GAPDH)

As PROTOCOL:ghosh-qpcr-pik3ca but with the HIF1A-specific primer pair (Suppl. Table 4).

PROTOCOL:ghosh-elisa-ppi3k-y508

Indirect ELISA for pPI3K p85alpha Tyr508

96-well plates coated in triplicate with 10 ug total protein per well, overnight at 4 C; blocked with 1% BSA in PBS for 1 h at 37 C; washed three times in 0.05% Tween-20/PBS with a 1-min soak. Primary antibody pPI3K p85alpha Tyr508 (Santa Cruz sc-12929, 1:1000) for 1 h at 37 C, then HRP-conjugated secondary (Santa Cruz, 1:2000) for 1 h at 37 C. TMB substrate (Merck Sigma Aldrich T444) in the dark, stopped with 1 M H2SO4, absorbance at 450 nm on an Infinite 200 PRO reader. Per-well normalization to total protein; phospho-signal specificity verified with phosphatase-treated lysates, and the phosphoprotein normalized to its total protein counterpart. Blank and no-primary-antibody wells subtracted. Intra-plate %CV 2-8%, inter-plate %CV 6-12%. Readouts are normalized relative units, not absolute concentrations.

PROTOCOL:ghosh-elisa-pakt-s473

Indirect ELISA for pAKT Ser473

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody pAKT Ser473 (Abclonal AP1208, 1:2000).

PROTOCOL:ghosh-elisa-pegfr-y1068

Indirect ELISA for pEGFR Tyr1068

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody pEGFR Tyr1068 (Abclonal AP0027, 1:2000).

PROTOCOL:ghosh-elisa-egfr

Indirect ELISA for total EGFR

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody EGFR (Santa Cruz sc-373746, 1:1000).

PROTOCOL:ghosh-elisa-pten

Indirect ELISA for PTEN

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody PTEN (Santa Cruz sc-7974, 1:1000).

PROTOCOL:ghosh-elisa-cyclind1

Indirect ELISA for Cyclin D1

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody Cyclin D1 (Santa Cruz sc-8396, 1:1000).

PROTOCOL:ghosh-elisa-bcl2

Indirect ELISA for Bcl-2

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody Bcl-2 (Santa Cruz sc-7382, 1:1000).

PROTOCOL:ghosh-elisa-bax

Indirect ELISA for BAX

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody BAX (Abclonal A11931, 1:2000).

PROTOCOL:ghosh-elisa-vegf

Indirect ELISA for VEGF

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody VEGF (Santa Cruz sc-57496, 1:1000).

PROTOCOL:ghosh-elisa-hif1a

Indirect ELISA for HIF-1alpha

As PROTOCOL:ghosh-elisa-ppi3k-y508 but with primary antibody HIF-1alpha (Abclonal A22041, 1:2000).