Airborne particulate matter increases MUC5AC expression by downregulating Claudin-1 expression in human airway cells.

NCI-H292 human lung mucoepidermoid carcinoma cell line

Normal human bronchial/nasal epithelial (NHNE) cells

Intracellular ROS production after CLB 2.0 in NCI-H292 cells

DCF flow cytometry time course in NCI-H292 cells exposed to CLB 2.0. ROS production peaked at 5 min and had already declined by 10 min, and it did not affect cell viability (CCK-8, NAC dose series). Cotreatment of CLB 2.0 with IL-6 (30 ng/ml) raised ROS production well above CLB 2.0 alone. The paper reports these panels as bar charts only (Fig. 3A-C) with no fluorescence values or fold changes, so no measurement slots are filled.

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Evidence

MUC5AC mRNA after CLB 2.0 with and without IL-6

qRT-PCR for MUC5AC transcript 24 h after treatment with CLB 2.0 (10 mg/ml), IL-6 (30 ng/ml), or both. MUC5AC gene expression increased significantly with the cotreatment of CLB 2.0 and IL-6 compared with CLB 2.0 alone, establishing that the IL-6 the cells secrete amplifies the mucin response rather than merely accompanying it. Reported as a bar chart (Fig. 1D) with significance marked only as P < 0.05; no fold-change values are tabulated, so muc5ac_mrna_expression is left unfilled.

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Evidence

MUC5AC mRNA under Claudin-1 overexpression or knockdown, NCI-H292 cells

NCI-H292 cells transfected with a wild-type Claudin-1 construct or Claudin-1-specific siRNA, then treated with CLB 2.0 (10 mg/ml) for 24 h before qRT-PCR for MUC5AC. Ectopic Claudin-1 dramatically inhibited MUC5AC gene expression while siRNA-Claudin-1 increased the CLB 2.0-induced MUC5AC, placing Claudin-1 as a negative regulator of the mucin response. This is the paper's central result: the loss of Claudin-1 caused by CLB 2.0 is itself what permits MUC5AC to rise. Reported as a bar chart (Fig. 2C) with significance marked only as P < 0.05; no fold-change values are tabulated.

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Evidence

MUC5AC mRNA with the ROS scavenger NAC, NCI-H292 cells

NCI-H292 cells pretreated 1 h with N-acetyl-cysteine (1 mM) and then treated with CLB 2.0 (10 mg/ml) for 24 h before qRT-PCR for MUC5AC. Inhibiting ROS production decreased MUC5AC gene expression (while restoring Claudin-1 and raising MUC1), making the mucin induction ROS-dependent. Reported as a bar chart (Fig. 3D, lower panel) with significance marked only as P < 0.05; no fold-change values are tabulated.

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Evidence

MUC5AC mRNA under ERK1/2 overexpression or knockdown, NCI-H292 cells

NCI-H292 cells transfected with a wild-type ERK1/2 construct or ERK1/2-specific siRNA, then treated with CLB 2.0 (10 mg/ml) for 24 h before qRT-PCR for MUC5AC. Wild-type ERK1/2 increased MUC5AC gene expression while siRNA-ERK1/2 dramatically decreased it, which is why the paper casts ERK1/2 as the switch between an inflamed state and homeostasis. Reported as a bar chart (Fig. 4C) with significance marked only as P < 0.05; no fold-change values are tabulated.

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Evidence

IL-6 secretion and overproduction after CLB 2.0

Cytokine array of conditioned medium, IL-6 Western blot of total cell lysates and IL-6 ELISA of supernatants, all 4 h after CLB 2.0. Of the cytokines and chemokines on the array, IL-6 secretion rose dramatically and dose-dependently, reaching its maximum at 4 h, and the Western blot and ELISA confirmed both secretion and overproduction in a time-dependent manner. Reported as array panels, blots and bar charts (Fig. 1A-C) with significance marked only as P < 0.05 and values described as means +/- SDs of three technical replicates from a single experiment; no concentrations are tabulated, so protein_level is left unfilled.

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Evidence

IL-6 expression with the ROS scavenger NAC, NCI-H292 cells

NCI-H292 cells pretreated 1 h with N-acetyl-cysteine (1 mM) and then treated with CLB 2.0 (10 mg/ml) for 24 h. Inhibiting ROS production decreased IL-6 expression, so the ROS burst is upstream of the cytokine rather than a consequence of it. Reported as a Western blot and bar chart (Fig. 3D) with no tabulated values.

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Evidence

Claudin-1, ZO-1 and E-cadherin protein after CLB 2.0 with and without IL-6, NCI-H292 cells

Western blot of tight junction proteins in NCI-H292 cells treated with CLB 2.0 (10 mg/ml) or CLB 2.0 plus IL-6 (30 ng/ml) for 24 h, with tubulin as loading control. Claudin-1 was inhibited by CLB 2.0 and the cotreatment inhibited it dramatically further. NULL RESULT: ZO-1 and E-cadherin were not changed by either treatment, so the effect is specific to Claudin-1 among the three tight junction proteins examined. A companion transfection arm (Fig. 2B) showed recombinant IL-6 with CLB 2.0 completely diminished Claudin-1 while siRNA-IL-6 only partially inhibited it, placing secreted IL-6 as a required co-factor. Blots are figure panels with no densitometry values, so protein_level is left unfilled.

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Evidence

Claudin-1 expression under ROS scavenging and ERK1/2 manipulation, NCI-H292 cells

Two rescue arms for the Claudin-1 loss. Pretreating NCI-H292 cells 1 h with N-acetyl-cysteine (1 mM) before CLB 2.0 increased Claudin-1 expression, and siRNA-ERK1/2 significantly increased CLB 2.0-induced Claudin-1 while wild-type ERK1/2 inhibited it. Together these place the Claudin-1 loss downstream of ROS and of ERK1/2. Reported as Western blots and bar charts (Figs. 3D and 4C) with no tabulated values.

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Evidence

MUC1 mRNA after CLB 2.0 with and without IL-6

qRT-PCR for MUC1 transcript 24 h after CLB 2.0 (10 mg/ml), IL-6 (30 ng/ml) or both. MUC1 gene expression decreased dramatically with the cotreatment of CLB 2.0 and IL-6. The paper treats MUC1 as the anti-inflammatory mucin that maintains homeostasis, the counterweight to inflammatory MUC5AC, which is why its fall marks a shift towards the inflamed state; the assay is therefore recorded against the airway inflammation key event rather than against mucin hypersecretion. Reported as a bar chart (Fig. 1E) with significance marked only as P < 0.05; no fold-change values are tabulated.

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Evidence

MUC1 mRNA under Claudin-1 overexpression, ROS scavenging and ERK1/2 knockdown, NCI-H292 cells

Three arms that all raise the anti-inflammatory mucin MUC1 in NCI-H292 cells treated with CLB 2.0 (10 mg/ml) for 24 h: overexpressed Claudin-1 increased CLB 2.0-induced MUC1 (and siRNA-Claudin-1 decreased it significantly), NAC pretreatment increased MUC1, and siRNA-ERK1/2 significantly increased MUC1 while wild-type ERK1/2 inhibited it. The paper reads this as Claudin-1 and the suppression of ROS/ERK1/2 restoring homeostasis rather than inflammation. Reported as bar charts (Figs. 2C, 3D, 4C) with no tabulated values.

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Evidence

ERK1/2, p38 and JNK phosphorylation after CLB 2.0, NCI-H292 cells

Western blot time course with phospho-specific antibodies in NCI-H292 cells treated with CLB 2.0. ERK1/2 phosphorylation reached a maximum at 15 min and had decreased by 30 min. NULL RESULT: p38 and JNK MAPKs were not phosphorylated, so the response is ERK-specific among the three MAPKs examined. NAC (1 mM, 1 h pretreatment) dramatically decreased the CLB 2.0-induced ERK1/2 activation, placing ERK1/2 downstream of the ROS burst. target_gene records MAPK3 (ERK1); the phospho-specific antibody detects both ERK1 and ERK2 and the paper reports them together as ERK1/2. Blots are figure panels with no densitometry values, so protein_level is left unfilled.

Exposure conditions

Outputs

Evidence

Increased oxidative stress leads to airway inflammation via IL-6 secretion

Increased oxidative stress leads to ERK1/2 activation

Airway inflammation leads to loss of epithelial barrier integrity via Claudin-1 downregulation

ERK1/2 activation leads to loss of epithelial barrier integrity via Claudin-1 downregulation

Loss of epithelial barrier integrity leads to mucin hypersecretion

Human cytokine array of conditioned medium

Cytokine assay performed with a Human Cytokine Array Panel A kit (R&D Systems) according to the manufacturer's instructions. Serum-starved cells were treated with CLB2 for 4 h; after treatment the supernatant was assayed according to the kit instructions. Run on cell culture medium in a dose-dependent manner and as a time course. Results are reported as array spot panels with no tabulated signal intensities.

Equipment: Human Cytokine Array Panel A kit (R&D Systems)

ELISA for IL-6 in cell supernatant

Cells were treated with CLB 2.0 for 4 h and their supernatants were collected; IL-6 levels in the cell supernatants were measured by ELISA. The ELISA kit and the purified IL-6 cytokine used as standard/treatment were purchased from R&D Systems. Results are reported as bar charts without tabulated concentrations.

Equipment: IL-6 ELISA kit (R&D Systems)

Western blot for IL-6

After treatment of the cells with CLB 2.0 for 4 h, total cell lysates were analyzed by Western blot analysis with specific anti-IL-6 antibody. No densitometry values are reported; the blots are shown as figure panels only.

Western blot for tight junction proteins Claudin-1, ZO-1 and E-cadherin

After the cells were treated with CLB 2.0 (10 mg/ml) or CLB 2.0 and IL-6 (30 ng/ml) for 24 h, Western blot analysis was performed with specific Claudin-1, ZO-1 and E-cadherin antibodies. Tubulin was used as the loading control. Blots are shown as figure panels with no densitometry values.

Western blot for MAPK phosphorylation (ERK1/2, p38, JNK)

Cells were treated with CLB 2.0 in a time-dependent manner and Western blots were performed with phospho-specific antibodies to detect MAPK activation (ERK1/2, p38 and JNK). Tubulin was used as the loading control. For the ROS-dependence arm, cells were treated with CLB 2.0 (10 mg/ml) or CLB 2.0 and NAC (1 mM; pretreatment for one hour) for 15 min before blotting for ERK1/2 phosphorylation. Blots are shown as figure panels with no densitometry values.

Real-time PCR for MUC5AC

Real-time PCR performed using a BioRad iQ iCycler Detection System (BioRad Laboratories; Hercules, CA) with iQ SYBR Green Supermix. Reactions were performed in a total volume of 20 ul, including 10 ul 2x SYBR Green PCR Master Mix, 300 nM of each primer, and 1 ul of the previously reverse-transcribed cDNA template. Total RNA was collected 24 h after treatment. The paper does not publish the MUC5AC primer sequences or the reference gene.

Equipment: BioRad iQ iCycler Detection System (BioRad Laboratories); iQ SYBR Green Supermix

Real-time PCR for MUC1

Real-time PCR performed using a BioRad iQ iCycler Detection System (BioRad Laboratories; Hercules, CA) with iQ SYBR Green Supermix. Reactions were performed in a total volume of 20 ul, including 10 ul 2x SYBR Green PCR Master Mix, 300 nM of each primer, and 1 ul of the previously reverse-transcribed cDNA template. Total RNA was collected 24 h after treatment. The paper does not publish the MUC1 primer sequences or the reference gene.

Equipment: BioRad iQ iCycler Detection System (BioRad Laboratories); iQ SYBR Green Supermix

Intracellular ROS measurement by DCF flow cytometry

Cells were pre-incubated in the presence of DCFH-DA (50 uM) for 30 min and then exposed to CLB 2.0 for the indicated times. Cell-associated DCF fluorescence levels were analyzed by flow cytometry. For the IL-6 cotreatment arm the cells were treated with CLB 2.0 (10 mg/ml) or CLB 2.0 and IL-6 (30 ng/ml) for 5 min before flow cytometry. Results are reported as bar charts with no tabulated fluorescence values.

Equipment: flow cytometer; DCFH-DA probe

Cell proliferation / viability assay (CCK-8)

Cells were pre-incubated in the presence of N-acetyl-cysteine (NAC) for 1 h in a dose-dependent manner and then treated with CLB 2.0 for 1 h. Cell proliferation assay was performed with CCK-8 (Dojindo; Rockville, MD).

Equipment: CCK-8 kit (Dojindo); microplate absorbance reader

Transient transfection with wild-type constructs or target-specific siRNA

Cells were transfected with either a construct driving the expression of the wild-type protein or an siRNA specific for the same target, then treated with CLB 2.0 (10 mg/ml) before harvest. Used for IL-6 (4 h before lysis, Claudin-1 blot), Claudin-1 (24 h before qRT-PCR for MUC5AC and MUC1) and ERK1/2 (24 h before qRT-PCR for MUC5AC and MUC1). The paper does not state the transfection reagent, vector backbone or siRNA sequences.