PM2.5 Upregulates the Expression of MUC5AC via the EGFR-PI3K Pathway in Human Sinonasal Epithelial Cells.

soma:jiao-hnec-control

Primary HNEC ALI culture, control subjects

soma:jiao-hnec-crswnp

Primary HNEC ALI culture, CRSwNP patients

GCM:jiao-muc5ac-mrna-control-24h

MUC5AC mRNA - control-subject HNECs, PM2.5 24 h

Real-time PCR for MUC5AC mRNA in ALI cultures from control subjects (n = 4). 100 ug/mL PM2.5 produced no significant change; 200 ug/mL PM2.5 increased MUC5AC mRNA to 2.13 +/- 0.37-fold that of medium-only cultures (full-text Results; significance reported only as p < 0.05). Values normalised to GAPDH and expressed relative to the medium-only control, which is therefore 1.0 by construction.

Exposure conditions

Outputs

Evidence

GCM:jiao-muc5ac-mrna-crswnp-24h

MUC5AC mRNA - CRSwNP HNECs, PM2.5 24 h

Real-time PCR for MUC5AC mRNA in ALI cultures from patients with chronic rhinosinusitis with nasal polyps (n = 6). 200 ug/mL PM2.5 increased MUC5AC mRNA to 1.71 +/- 0.41-fold that of medium-only cultures (full-text Results; p < 0.05). Values normalised to GAPDH and expressed relative to the medium-only control.

Exposure conditions

Outputs

Evidence

GCM:jiao-muc5ac-positive-control-24h

MUC5AC-positive cells - control-subject HNECs, PM2.5 24 h

Immunofluorescence quantification of MUC5AC-positive cells as a percentage of total cells in ALI cultures from control subjects (n = 4), reported as the relative change from medium-only cultures. 100 ug/mL PM2.5 produced a non-significant increasing trend (1.16 +/- 0.21-fold); 200 ug/mL PM2.5 increased MUC5AC-positive cells 2.29 +/- 0.41-fold (full-text Results; p < 0.05). beta-tubulin IV-positive cell percentage was unchanged in the same cultures (recorded separately in ciliary_function_assays). SLOT NOTE: the paper frames this immunocytochemistry readout as MUC5AC protein expression, but the measurement is a stained-cell fraction, so it is recorded in goblet_cell_percentage rather than muc5ac_protein_expression; this study ran no Western blot or ELISA for MUC5AC protein (the Western arm is p-PI3K/p-AKT/p-ERK only).

Exposure conditions

Outputs

Evidence

GCM:jiao-muc5ac-positive-crswnp-24h

MUC5AC-positive cells - CRSwNP HNECs, PM2.5 24 h

Immunofluorescence quantification of MUC5AC-positive cells in ALI cultures from CRSwNP patients (n = 6), reported as the relative change from medium-only cultures. 100 ug/mL PM2.5 gave a non-significant trend (1.13 +/- 0.45-fold); 200 ug/mL PM2.5 increased MUC5AC-positive cells 2.68 +/- 0.67-fold (full-text Results; p < 0.05). SLOT NOTE: the paper frames this immunocytochemistry readout as MUC5AC protein expression, but the measurement is a stained-cell fraction, so it is recorded in goblet_cell_percentage rather than muc5ac_protein_expression; this study ran no Western blot or ELISA for MUC5AC protein (the Western arm is p-PI3K/p-AKT/p-ERK only).

Exposure conditions

Outputs

Evidence

GCM:jiao-muc5ac-positive-ag1478-crswnp

MUC5AC-positive cells - EGFR inhibitor AG1478 + PM2.5, CRSwNP HNECs

CRSwNP-derived ALI cultures (n = 4) pretreated for 30 min with the EGFR inhibitor AG1478 (10 uM) before 200 ug/mL PM2.5 for 24 h. MUC5AC-positive cell percentages were 2.51 +/- 0.44-fold (PM2.5 alone) and 1.50 +/- 0.21-fold (AG1478 + PM2.5) relative to medium-only cells (full-text Results; p < 0.05). AG1478 alone did not affect basal MUC5AC protein expression. The inhibitor was added 30 min before PM2.5 and remained until the end of the experiment, so the inhibitor exposure condition carries the full 24 h duration. SLOT NOTE: the paper frames this immunocytochemistry readout as MUC5AC protein expression, but the measurement is a stained-cell fraction, so it is recorded in goblet_cell_percentage rather than muc5ac_protein_expression; this study ran no Western blot or ELISA for MUC5AC protein (the Western arm is p-PI3K/p-AKT/p-ERK only).

Exposure conditions

Outputs

Evidence

GE:jiao-spdef-mrna-control-24h

SPDEF mRNA - control-subject HNECs, PM2.5 24 h

SPDEF (SAM-pointed domain-containing ETS transcription factor) is the key transcription factor for goblet cell differentiation and mucus production. 200 ug/mL PM2.5 for 24 h increased SPDEF mRNA to 1.70 +/- 0.26-fold that of medium-only cultures from control subjects (n = 4; full-text Results; p < 0.05).

Exposure conditions

Outputs

Evidence

GE:jiao-spdef-mrna-crswnp-24h

SPDEF mRNA - CRSwNP HNECs, PM2.5 24 h

200 ug/mL PM2.5 for 24 h increased SPDEF mRNA to 2.32 +/- 1.55-fold that of medium-only cultures from CRSwNP patients (n = 6; full-text Results; p < 0.05).

Exposure conditions

Outputs

Evidence

GE:jiao-hbegf-mrna-control-24h

HB-EGF mRNA - control-subject HNECs, PM2.5 24 h

Heparin-binding EGF-like growth factor mRNA rose to 1.44 +/- 0.15-fold that of medium-only cultures from control subjects after 200 ug/mL PM2.5 for 24 h (n = 4; full-text Results; p < 0.05). HB-EGF protein measured by ELISA was likewise increased.

Exposure conditions

Outputs

Evidence

GE:jiao-hbegf-mrna-crswnp-24h

HB-EGF mRNA - CRSwNP HNECs, PM2.5 24 h

Heparin-binding EGF-like growth factor mRNA rose to 1.56 +/- 0.56-fold that of medium-only cultures from CRSwNP patients after 200 ug/mL PM2.5 for 24 h (n = 6; full-text Results; p < 0.05).

Exposure conditions

Outputs

Evidence

GE:jiao-areg-mrna-control-24h

Amphiregulin mRNA - control-subject HNECs, PM2.5 24 h

Amphiregulin mRNA rose to 1.42 +/- 0.14-fold that of medium-only cultures from control subjects after 200 ug/mL PM2.5 for 24 h (n = 4; full-text Results; p < 0.05). AREG protein by ELISA was also significantly increased.

Exposure conditions

Outputs

Evidence

GE:jiao-areg-mrna-crswnp-24h

Amphiregulin mRNA - CRSwNP HNECs, PM2.5 24 h

Amphiregulin mRNA rose to 1.23 +/- 0.25-fold that of medium-only cultures from CRSwNP patients after 200 ug/mL PM2.5 for 24 h (n = 6; full-text Results; p < 0.05).

Exposure conditions

Outputs

Evidence

GE:jiao-ereg-mrna-control-24h

Epiregulin mRNA - control-subject HNECs, PM2.5 24 h

Epiregulin mRNA rose to 1.46 +/- 0.31-fold that of medium-only cultures from control subjects after 200 ug/mL PM2.5 for 24 h (n = 4; full-text Results; p < 0.05). EREG protein could not be confirmed: its concentration was below the detection range of the ELISA kit. EGF, TGF-alpha and neuregulin mRNA were not significantly elevated.

Exposure conditions

Outputs

Evidence

GE:jiao-ereg-mrna-crswnp-24h

Epiregulin mRNA - CRSwNP HNECs, PM2.5 24 h

Epiregulin mRNA rose to 1.94 +/- 1.03-fold that of medium-only cultures from CRSwNP patients after 200 ug/mL PM2.5 for 24 h (n = 6; full-text Results; p < 0.05).

Exposure conditions

Outputs

Evidence

GE:jiao-hbegf-protein-control-24h

HB-EGF protein (ELISA) - control-subject HNECs, PM2.5 24 h

ELISA for heparin-binding EGF-like growth factor protein in ALI cultures from control subjects (n = 4) treated with 200 ug/mL PM2.5 for 24 h. HB-EGF protein was increased relative to medium-only cultures (Fig. 4; p < 0.05), matching the HB-EGF mRNA rise recorded in GE:jiao-hbegf-mrna-control-24h. Values are expressed as pg per mg total cell culture protein (Bradford assay). The paper shows these data graphically only, so neither output carries a numeric protein_level; COMP:jiao-hbegf-protein-control records the direction without a magnitude.

Exposure conditions

Outputs

Evidence

GE:jiao-hbegf-protein-crswnp-24h

HB-EGF protein (ELISA) - CRSwNP HNECs, PM2.5 24 h

ELISA for heparin-binding EGF-like growth factor protein in ALI cultures from CRSwNP patients (n = 4; the ELISA arm used four CRSwNP-derived cultures, not the six of the mRNA arm) treated with 200 ug/mL PM2.5 for 24 h. HB-EGF protein was increased relative to medium-only cultures (Fig. 4; p < 0.05). Values are expressed as pg per mg total cell culture protein (Bradford assay). The paper shows these data graphically only, so neither output carries a numeric protein_level; COMP:jiao-hbegf-protein-crswnp records the direction without a magnitude.

Exposure conditions

Outputs

Evidence

GE:jiao-areg-protein-control-24h

Amphiregulin protein (ELISA) - control-subject HNECs, PM2.5 24 h

ELISA for amphiregulin protein in ALI cultures from control subjects (n = 4) treated with 200 ug/mL PM2.5 for 24 h. AREG protein was increased relative to medium-only cultures (Fig. 4; p < 0.05), matching the AREG mRNA rise recorded in GE:jiao-areg-mrna-control-24h. Values are expressed as pg per mg total cell culture protein (Bradford assay). The paper shows these data graphically only, so neither output carries a numeric protein_level; COMP:jiao-areg-protein-control records the direction without a magnitude.

Exposure conditions

Outputs

Evidence

GE:jiao-areg-protein-crswnp-24h

Amphiregulin protein (ELISA) - CRSwNP HNECs, PM2.5 24 h

ELISA for amphiregulin protein in ALI cultures from CRSwNP patients (n = 4; the ELISA arm used four CRSwNP-derived cultures, not the six of the mRNA arm) treated with 200 ug/mL PM2.5 for 24 h. AREG protein was increased relative to medium-only cultures (Fig. 4; p < 0.05). Values are expressed as pg per mg total cell culture protein (Bradford assay). The paper shows these data graphically only, so neither output carries a numeric protein_level; COMP:jiao-areg-protein-crswnp records the direction without a magnitude.

Exposure conditions

Outputs

Evidence

GE:jiao-tgfa-protein-control-24h

TGF-alpha protein (ELISA) - control-subject HNECs, PM2.5 24 h (no change)

NULL RESULT. ELISA for transforming growth factor-alpha protein in ALI cultures from control subjects (n = 4) treated with 200 ug/mL PM2.5 for 24 h. There was no significant increase in TGF-alpha protein (Fig. 4), the one EGFR ligand of the three assayed whose protein did not respond; TGF-alpha mRNA was likewise not significantly elevated, as this file's EGFR ligand mRNA records already note. The paper shows these data graphically only and reports no significant difference, so no values are recorded and no response_comparison is derived, following the convention used for this file's other null results.

Exposure conditions

Outputs

Evidence

GE:jiao-tgfa-protein-crswnp-24h

TGF-alpha protein (ELISA) - CRSwNP HNECs, PM2.5 24 h (no change)

NULL RESULT. ELISA for transforming growth factor-alpha protein in ALI cultures from CRSwNP patients (n = 4) treated with 200 ug/mL PM2.5 for 24 h. There was no significant increase in TGF-alpha protein (Fig. 4). The paper states the null result for TGF-alpha without separating the donor groups, and Figure 4 carries no significance marker on either TGF-alpha bar. The data are shown graphically only, so no values are recorded and no response_comparison is derived.

Exposure conditions

Outputs

Evidence

GE:jiao-muc5ac-mrna-ly294002-crswnp

MUC5AC mRNA - PI3K inhibitor LY294002 + PM2.5, CRSwNP HNECs

CRSwNP-derived ALI cultures (n = 4) pretreated for 30 min with the PI3K inhibitor LY294002 (25 uM) before 200 ug/mL PM2.5 for 24 h. MUC5AC mRNA expression was significantly blocked by LY294002 in PM2.5-stimulated cells (Fig. 6; p < 0.05); LY294002 alone did not affect basal MUC5AC expression. The paper reports this result graphically only (Fig. 6), so neither treated group carries a fold-change value; COMP:jiao-muc5ac-mrna-ly294002 records the direction of the attenuation without a magnitude. LY294002 was added 30 min before PM2.5 and remained until the end of the experiment, so its exposure condition carries the full 24 h duration; the 200 ug/mL PM2.5 co-exposure is listed as its own condition on this assay.

Exposure conditions

Outputs

Evidence

EGFR:jiao-pi3k-akt-erk-30min

PI3K-AKT/ERK activation after PM2.5, CRSwNP HNECs

Western blot of CRSwNP-derived ALI cultures (n = 4) treated with 200 ug/mL PM2.5 for 30 min. p-PI3K, p-AKT and p-ERK were all significantly increased while total PI3K, AKT and ERK protein was unchanged (Fig. 5; p < 0.05). The paper reports the densitometry graphically only, so no numeric phosphorylation values are recorded. EGFR phosphorylation itself was not measured: EGFR involvement rests on the ligand data and on reversal by the EGFR inhibitor AG1478.

Exposure conditions

Outputs

Evidence

FOXJ:jiao-foxj1-mrna-control-24h

FOXJ1 mRNA - control-subject HNECs, PM2.5 24 h (no change)

NULL RESULT. Real-time PCR for FOXJ1 in ALI cultures from control subjects (n = 4). Neither 100 nor 200 ug/mL PM2.5 for 24 h altered FOXJ1 mRNA expression; the same was true of GMNC, MCIDAS and DNAI2. No fold-change values are recorded because the paper reports these data graphically only and states no significant difference. The authors attribute the absent effect to the short exposure time or the low PM2.5 concentrations.

Exposure conditions

Outputs

Evidence

FOXJ:jiao-foxj1-mrna-crswnp-24h

FOXJ1 mRNA - CRSwNP HNECs, PM2.5 24 h (no change)

NULL RESULT. Real-time PCR for FOXJ1 in ALI cultures from CRSwNP patients (n = 6). Neither 100 nor 200 ug/mL PM2.5 for 24 h altered FOXJ1 mRNA expression; the same was true of GMNC, MCIDAS and DNAI2. No fold-change values are recorded because the paper reports these data graphically only and states no significant difference. The authors attribute the absent effect to the short exposure time or the low PM2.5 concentrations.

Exposure conditions

Outputs

Evidence

CILIA:jiao-btubulin-positive-control-24h

Ciliated cell coverage (beta-tubulin IV-positive cells) - control-subject HNECs, PM2.5 24 h (no change)

NULL RESULT. Immunofluorescence quantification of beta-tubulin IV-positive cells as a percentage of total cells in ALI cultures from control subjects (n = 4). No significant change in ciliated cell coverage was observed at either 100 or 200 ug/mL PM2.5 for 24 h, in contrast to the MUC5AC-positive cell increase measured in the same stained cultures. The paper reports these data graphically only, so no values are recorded.

Exposure conditions

Outputs

Evidence

CILIA:jiao-btubulin-positive-crswnp-24h

Ciliated cell coverage (beta-tubulin IV-positive cells) - CRSwNP HNECs, PM2.5 24 h (no change)

NULL RESULT. Immunofluorescence quantification of beta-tubulin IV-positive cells as a percentage of total cells in ALI cultures from CRSwNP patients (n = 6). No significant change in ciliated cell coverage was observed at either 100 or 200 ug/mL PM2.5 for 24 h, in contrast to the MUC5AC-positive cell increase measured in the same stained cultures. The paper reports these data graphically only, so no values are recorded.

Exposure conditions

Outputs

Evidence

COMP:jiao-muc5ac-mrna-control

MUC5AC mRNA, 200 ug/mL PM2.5 vs medium (control donors)

COMP:jiao-muc5ac-mrna-crswnp

MUC5AC mRNA, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

COMP:jiao-muc5ac-positive-control-200

MUC5AC-positive cells, 200 ug/mL PM2.5 vs medium (control donors)

COMP:jiao-muc5ac-positive-control-100

MUC5AC-positive cells, 100 ug/mL PM2.5 vs medium (control donors, trend only)

Reported as an increasing trend; the paper does not mark this comparison as significant.

COMP:jiao-muc5ac-positive-crswnp-200

MUC5AC-positive cells, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

COMP:jiao-muc5ac-positive-crswnp-100

MUC5AC-positive cells, 100 ug/mL PM2.5 vs medium (CRSwNP donors, trend only)

Reported as an increasing trend; the paper does not mark this comparison as significant.

COMP:jiao-muc5ac-positive-ag1478-pm25

MUC5AC-positive cells, PM2.5 200 ug/mL vs medium (CRSwNP donors, inhibitor experiment)

The PM2.5-alone arm of the AG1478 experiment, kept against the same medium-only anchor as the inhibitor arm so the two are directly comparable. A separate record from COMP:jiao-muc5ac-positive-crswnp-200 because it comes from the inhibitor experiment (n = 4), not the dose-response experiment (n = 6).

COMP:jiao-muc5ac-positive-ag1478

MUC5AC-positive cells, AG1478 + PM2.5 vs medium (CRSwNP donors)

EGFR inhibition arm. The paper states 2.51 +/- 0.44-fold for PM2.5 alone and 1.50 +/- 0.21-fold for AG1478-pretreated cells, both relative to medium-only cells; this record keeps the reported AG1478 + PM2.5 value against the same medium-only anchor rather than computing the attenuation.

COMP:jiao-spdef-mrna-control

SPDEF mRNA, 200 ug/mL PM2.5 vs medium (control donors)

COMP:jiao-spdef-mrna-crswnp

SPDEF mRNA, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

COMP:jiao-hbegf-mrna-control

HB-EGF mRNA, 200 ug/mL PM2.5 vs medium (control donors)

COMP:jiao-hbegf-mrna-crswnp

HB-EGF mRNA, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

COMP:jiao-areg-mrna-control

AREG mRNA, 200 ug/mL PM2.5 vs medium (control donors)

COMP:jiao-areg-mrna-crswnp

AREG mRNA, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

COMP:jiao-ereg-mrna-control

EREG mRNA, 200 ug/mL PM2.5 vs medium (control donors)

COMP:jiao-ereg-mrna-crswnp

EREG mRNA, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

COMP:jiao-hbegf-protein-control

HB-EGF protein, 200 ug/mL PM2.5 vs medium (control donors)

Direction only: PM2.5 increased HB-EGF protein in control-derived cultures (Fig. 4, p < 0.05), but the paper reports the ELISA values graphically without a mean or SEM, so no change_value is recorded.

COMP:jiao-hbegf-protein-crswnp

HB-EGF protein, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

Direction only: PM2.5 increased HB-EGF protein in CRSwNP-derived cultures (Fig. 4, p < 0.05), with no numeric value reported.

COMP:jiao-areg-protein-control

AREG protein, 200 ug/mL PM2.5 vs medium (control donors)

Direction only: PM2.5 increased amphiregulin protein in control-derived cultures (Fig. 4, p < 0.05), with no numeric value reported.

COMP:jiao-areg-protein-crswnp

AREG protein, 200 ug/mL PM2.5 vs medium (CRSwNP donors)

Direction only: PM2.5 increased amphiregulin protein in CRSwNP-derived cultures (Fig. 4, p < 0.05), with no numeric value reported.

COMP:jiao-muc5ac-mrna-ly294002

MUC5AC mRNA, LY294002 + PM2.5 vs PM2.5 alone (CRSwNP donors)

PI3K inhibition attenuates the PM2.5-induced rise in MUC5AC mRNA. Direction only: the paper reports this contrast graphically (Fig. 6) and states significance as p < 0.05 without a fold-change value, so no change_value is recorded. The reference group here is PM2.5 alone, not medium.

COMP:jiao-muc5ac-positive-ag1478-attenuation

MUC5AC-positive cells, AG1478 + PM2.5 vs PM2.5 alone (CRSwNP donors)

EGFR inhibition attenuates the PM2.5-induced rise in MUC5AC-positive cells (2.51 +/- 0.44-fold vs medium for PM2.5 alone, 1.50 +/- 0.21-fold vs medium with AG1478). Direction only: the paper reports both values against medium and calls the attenuation significant, but never states the PM2.5-vs-inhibitor ratio, so no change_value is computed here. The reference group is PM2.5 alone, not medium.

KER:jiao-egfr-to-mucin

EGFR activation leads to mucin hypersecretion

Central mechanism of Jiao 2022: PM2.5 raises EGFR ligand expression (HB-EGF, AREG, EREG) and activates PI3K-AKT/ERK, and blocking either EGFR (AG1478) or PI3K (LY294002) attenuates the PM2.5-induced rise in MUC5AC mRNA and protein in human nasal epithelial cells.

KER:jiao-egfr-to-goblet

EGFR activation leads to goblet cell hyperplasia

The percentage of MUC5AC-positive (goblet) cells rose with PM2.5 and was reduced by EGFR inhibition with AG1478, linking EGFR activation to the goblet cell phenotype in nasal epithelium.

PROTOCOL:jiao-ali-001

Primary HNEC air-liquid interface culture

Nasal mucosal tissue dissociated in 0.1% protease type XIV in DMEM overnight at 4C. Cells seeded at 150,000 per well onto human placental collagen-precoated 6.5-mm Transwell polyester membranes (0.4 um pore, Costar/Corning). Submerged culture in bronchial epithelial growth medium:DMEM (1:1); ALI established at confluence by removing apical medium, basolateral ALI medium containing 50 nM all-trans retinoic acid, changed every other day for 14 days until differentiated. PM2.5 added to the apical compartment; control cultures received ALI culture medium only.

Equipment: Transwell polyester membrane inserts, 6.5 mm, 0.4 um pore (Costar, Corning); Human placental collagen coating; Bronchial epithelial growth medium:DMEM (1:1) with 50 nM all-trans retinoic acid

PROTOCOL:jiao-qpcr-muc5ac

Real-time PCR for MUC5AC

ALI cultures lysed in RLT buffer after 24 h PM2.5; total RNA extracted with RNeasy mini kit (Qiagen). Reverse transcription with PrimeScript RT Master Mix (TaKaRa). SYBR Green assay on ABI7500 Real-Time PCR Detection System (Applied Biosystems). GAPDH endogenous reference; relative expression by the comparative cycle threshold method.

Equipment: ABI7500 Real-Time PCR Detection System (Applied Biosystems); RNeasy mini kit (Qiagen); PrimeScript RT Master Mix (TaKaRa Biotechnology)

PROTOCOL:jiao-qpcr-spdef

Real-time PCR for SPDEF

ALI cultures lysed in RLT buffer after 24 h PM2.5; total RNA extracted with RNeasy mini kit (Qiagen). Reverse transcription with PrimeScript RT Master Mix (TaKaRa). SYBR Green assay on ABI7500 Real-Time PCR Detection System (Applied Biosystems). GAPDH endogenous reference; relative expression by the comparative cycle threshold method.

Equipment: ABI7500 Real-Time PCR Detection System (Applied Biosystems); RNeasy mini kit (Qiagen); PrimeScript RT Master Mix (TaKaRa Biotechnology)

PROTOCOL:jiao-qpcr-hbegf

Real-time PCR for HB-EGF

ALI cultures lysed in RLT buffer after 24 h PM2.5; total RNA extracted with RNeasy mini kit (Qiagen). Reverse transcription with PrimeScript RT Master Mix (TaKaRa). SYBR Green assay on ABI7500 Real-Time PCR Detection System (Applied Biosystems). GAPDH endogenous reference; relative expression by the comparative cycle threshold method.

Equipment: ABI7500 Real-Time PCR Detection System (Applied Biosystems); RNeasy mini kit (Qiagen); PrimeScript RT Master Mix (TaKaRa Biotechnology)

PROTOCOL:jiao-qpcr-areg

Real-time PCR for AREG

ALI cultures lysed in RLT buffer after 24 h PM2.5; total RNA extracted with RNeasy mini kit (Qiagen). Reverse transcription with PrimeScript RT Master Mix (TaKaRa). SYBR Green assay on ABI7500 Real-Time PCR Detection System (Applied Biosystems). GAPDH endogenous reference; relative expression by the comparative cycle threshold method.

Equipment: ABI7500 Real-Time PCR Detection System (Applied Biosystems); RNeasy mini kit (Qiagen); PrimeScript RT Master Mix (TaKaRa Biotechnology)

PROTOCOL:jiao-qpcr-ereg

Real-time PCR for EREG

ALI cultures lysed in RLT buffer after 24 h PM2.5; total RNA extracted with RNeasy mini kit (Qiagen). Reverse transcription with PrimeScript RT Master Mix (TaKaRa). SYBR Green assay on ABI7500 Real-Time PCR Detection System (Applied Biosystems). GAPDH endogenous reference; relative expression by the comparative cycle threshold method.

Equipment: ABI7500 Real-Time PCR Detection System (Applied Biosystems); RNeasy mini kit (Qiagen); PrimeScript RT Master Mix (TaKaRa Biotechnology)

PROTOCOL:jiao-qpcr-foxj1

Real-time PCR for FOXJ1

ALI cultures lysed in RLT buffer after 24 h PM2.5; total RNA extracted with RNeasy mini kit (Qiagen). Reverse transcription with PrimeScript RT Master Mix (TaKaRa). SYBR Green assay on ABI7500 Real-Time PCR Detection System (Applied Biosystems). GAPDH endogenous reference; relative expression by the comparative cycle threshold method.

Equipment: ABI7500 Real-Time PCR Detection System (Applied Biosystems); RNeasy mini kit (Qiagen); PrimeScript RT Master Mix (TaKaRa Biotechnology)

PROTOCOL:jiao-icc-muc5ac

Immunocytochemical staining for MUC5AC

ALI cultures fixed in 50:50 methanol:acetone, permeabilized with 0.3% Triton X-100, blocked with 5% skim milk, incubated overnight at 4C with anti-beta-tubulin IV (1:500, Sigma-Aldrich) and anti-MUC5AC (1:200, Abcam). Secondary antibodies labelled with fluorescein isothiocyanate (1:500, Invitrogen) and rhodamine (1:500, Invitrogen). Nuclei counterstained with 4,6-diamidino-2-phenylindole. Imaged on an Olympus IX81 confocal microscope; positive cells expressed as a percentage of total cells.

Equipment: Olympus IX81 confocal microscope; anti-beta-tubulin IV (Sigma-Aldrich); anti-MUC5AC (Abcam)

PROTOCOL:jiao-icc-btub

Immunocytochemical staining for beta-tubulin IV

ALI cultures fixed in 50:50 methanol:acetone, permeabilized with 0.3% Triton X-100, blocked with 5% skim milk, incubated overnight at 4C with anti-beta-tubulin IV (1:500, Sigma-Aldrich) and anti-MUC5AC (1:200, Abcam). Secondary antibodies labelled with fluorescein isothiocyanate (1:500, Invitrogen) and rhodamine (1:500, Invitrogen). Nuclei counterstained with 4,6-diamidino-2-phenylindole. Imaged on an Olympus IX81 confocal microscope; positive cells expressed as a percentage of total cells.

Equipment: Olympus IX81 confocal microscope; anti-beta-tubulin IV (Sigma-Aldrich); anti-MUC5AC (Abcam)

PROTOCOL:jiao-elisa-hbegf

ELISA for HB-EGF protein

ALI cultures treated with PM2.5 for 24 h assayed with a commercial HB-EGF ELISA kit (Cloud Clone Corp., Wuhan). Absorbance read at 450 nm. Protein concentration of each culture determined by Bradford assay.

Equipment: ELISA plate reader (450 nm); HB-EGF ELISA kit (Cloud Clone Corp., Wuhan)

PROTOCOL:jiao-elisa-areg

ELISA for AREG protein

ALI cultures treated with PM2.5 for 24 h assayed with a commercial AREG ELISA kit (Cloud Clone Corp., Wuhan). Absorbance read at 450 nm. Protein concentration of each culture determined by Bradford assay.

Equipment: ELISA plate reader (450 nm); AREG ELISA kit (Cloud Clone Corp., Wuhan)

PROTOCOL:jiao-elisa-tgfa

ELISA for TGF-alpha protein

ALI cultures treated with PM2.5 for 24 h assayed with a commercial TGF-alpha ELISA kit (Cloud Clone Corp., Wuhan). Absorbance read at 450 nm. Protein concentration of each culture determined by Bradford assay.

Equipment: ELISA plate reader (450 nm); TGF-alpha ELISA kit (Cloud Clone Corp., Wuhan)

PROTOCOL:jiao-wb-001

Western blot for PI3K, AKT and ERK phosphorylation

ALI cultures treated with PM2.5 for 30 min harvested in RIPA lysis buffer with protease and phosphatase inhibitors. Equal protein separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, blocked in 5% BSA, incubated overnight at 4C with anti-p-PI3K (1:1,000), anti-PI3K (1:2,000), anti-p-AKT (1:1,000), anti-AKT (1:2,000), anti-p-ERK (1:1,000), anti-ERK (1:2,000) (Cell Signaling Technology) and anti-GAPDH (1:10,000, Abcam). Electrogenerated chemiluminescence detection; bands analysed in ImageJ.

Equipment: SDS-PAGE and polyvinylidene fluoride transfer apparatus; Electrogenerated chemiluminescence detection system; ImageJ densitometry software