Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: (a & b) Representative I sc tracings of CFTR-dependent short-circuit currents ( I sc ) were measured in Ussing chambers. (c) Representative Western blot images of total cellular CFTR and β2AR expression levels in treated dBCi cells after Ussing chamber analyses in ( a ). (d) Densitometric analyses showing B[a]P-induced reduction of both β2AR and CFTR expression in a dose-dependent manner. (e) The Pearson correlation coefficient (r) showing a significant positive relationship between B[a]P-inhibited β2AR and CFTR expression. All data were obtained from three independent experiments. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P treatments to the DMSO control; * p < 0.01.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: Both apical and basolateral sides of polarized 16HBE14o-cells (ALI for 8-10 days) were incubated with different concentrations of B[a]P. Treated media from the apical sides were removed after 6-hr incubation, and cell treatment continued further for 18 hrs under ALI conditions. ( a ) Representative I sc tracings of CFTR-dependent short-circuit currents (I sc ) measured in Ussing chambers as the changes in response to Amiloride, IBMX/Forskolin, and CFTRinh-172. (b) Summary of changes in I sc is shown. Data represent the mean of three independent experiments, and each experiment was performed in duplication (n = 3). Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P treatments to the DMSO control; * p < 0.05; ** p < 0.01. ( c ) Representative Western blot images of β2AR and CFTR expression levels in treated cells after Ussing chamber analyses in ( a ).

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Incubation, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: Cells were treated with various concentrations of PM2.5 for 24 hr. ( a ) Cell viability was assessed with MTT assay. ( b ) IL-6 and IL-8 from the basolateral sides. ( c ) Representative Western blot images showing CFTR, β2AR, phospho-AKT (Ser473), AKT, phospho-NF-κB p65 (Ser536), NF-κB p65, and TRPC6 expression. All data are presented as the mean ± SD (n = 4). Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P or PM 2.5 treatments to the DMSO control; *, p < 0.05; ¥, p < 0.01; NS, no significance.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: MTT Assay, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: Both apical and basolateral sides of polarized 16HBE14o-cells (ALI for 8-10 days) were incubated with different concentrations of PM 2.5. Treated media from the apical sides were removed after 6-hr incubation, and cell treatment continued further for 18 hrs under ALI conditions. ( a ) Representative I sc tracings of CFTR-dependent short-circuit currents (I sc ) measured in Ussing chambers as the changes in response to Amiloride, IBMX/Forskolin, and CFTRinh-172. (b) Summary of changes in I sc is shown. Data represent the mean of three independent experiments, and each experiment was performed in duplication (n = 6). Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P treatments to the DMSO control; ¥, p < 0.05; *, p < 0.01. ( c ) Representative Western blot images of β2AR and CFTR expression levels in treated cells after Ussing chamber analyses in ( a). All Western analyses represent the results of three independent experiments, and β-actin was used for equal loading of protein.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Incubation, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: (a) Representative Western blot images of cell surface CFTR and β2AR expression in cells treated with DMSO or various concentrations of B[a]P. (b & c) Recycling assay of endocytosis CFTR and β2AR were analyzed by Western blot. Representative Western blot images of biotinylated CFTR and β2AR expression are shown. Cell samples treated without or with GSH ( lanes 1 and 2 ) were used as positive controls for biotinylation and GSH stripping processes, respectively. (d) A representative Western blot image showing ubiquitination of CFTR immunoprecipitates from cell lysates treated with different concentrations of B[a]P in the presence of 20 µM MG 132. All Western blot analyses represent the results of three independent experiments.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Western Blot, Expressing, Stripping Membranes, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: ALI cells were treated with DMSO, 100 ng/mL PM 2.5, 10 µM LY 294002 ± PM 2.5, or 10 µM LY 303511 ± PM 2.5 for 24 hrs. ( a ) IL-6 and IL-8 from the basolateral sides were determined using the DuoSet® ELISA kits. Data are presented as the mean ± SD (n=3). Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing all treatments to the DMSO control, or PM 2.5 to PM 2.5 ± LY 294002 or LY 303511; *, p < 0.01. ( b ) Representative Western blot images showing CFTR and β-actin expression. β-actin was used to ensure equal loading of protein. All data were obtained from three independent experiments, and each experiment was analyzed in duplication.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: ( a ) Fluo-4-AM fluorescence intensity traces obtained 24 hours after treatment with B[a]P and stimulated with assay buffer containing 1 mM Ca +2 . ( b ) Corresponding histograms summarize the mean changes in RFU (F 1 – F 0 ) after stimulation by 1 mM Ca +2 . ( c & d ) Representative Western blot images of β2AR and TRPC6 expression levels in treated cell in ( a ) and respective densitometric analyses. ( e ) The Pearson correlation coefficient (r) showing a significant negative relationship between β2AR and TRPC6 expression caused by B[a]P treatment. ( f ) Fluo-4AM fluorescence intensity traces in 16HBE14o-cells were obtained before and after addition of 2 mM Ca +2 in the presence or absence of 5µM BI 749327. ( g ) Corresponding histograms summarize the mean changes in RFU (F 1 – F 0 ) after stimulation by 2 mM Ca +2 . ( h ) Representative western blotting analyses of CFTR expression in corresponding treated cells. All data represent the mean of three independent experiments. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P ± BI 749327 treatments to the DMSO control; ¥, p<0.05, * p < 0.01.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Fluorescence, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: ( a ) After 24 hr treatment, fluorescence intensity traces obtained using the fluo-4 AM when DMSO- or PM 2.5-treated 16HBE14o-cellswere stimulated with assay buffer containing 2 mM Ca. Each trace is the mean of three independent experiments with standard deviations (vertical bars). ( b ) Corresponding histograms summarize the mean area under the curve (AUC) after stimulation by 2 mM Ca. ( c ) Representative Western blot images of CFTR, β2AR and TRPC6 expression levels in treated cell in ( a ). All data were obtained from four independent experiments. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the PM 2.5 treatments to the DMSO control; ¥, p < 0.05; *, p < 0.01.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Fluorescence, Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: ( a ) The lung parenchyma from two ferrets were sliced into several sections as shown. Arrows indicate the tracheobronchial tubes. All tissue sections were incubating with DMSO (control), 10 µM B[a]P, or 100 ng/mL PM 2.5 in Pneumacult-EX Plus Basal media for 24 hrs. ( b ) IL 6 secretion was determined using the ferret ELISA kit. Data represent the mean of 6 independent sections. Statistical p values were determined with a one-way ANOVA, followed by the Tukey post-hoc test comparing the B[a]P or PM 2.5 treatments to the DMSO control; ¥, p < 0.05 (n = 6). ( c ) Representative western blotting images show reduction of CFTR, and β2AR and elevation of TRPC6 expression in explants under these treatments.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: PM2.5 toxin benzo[a]pyrene induces life-limiting inflammation and oxidative stress in the airway by up-regulation of TRPC6 and inactivation of β2AR/CFTR signaling

doi: 10.64898/2026.04.21.719931

Figure Lengend Snippet: PM 2.5 or the PM 2.5 toxin B[a]P initially acts as an irreversible β2AR agonist to hyperactivate the PI3K signaling pathway. PLC, acting on PIP2, releases DAG. DAG activates the TRPC6 channel to bring extracellular Ca +2 into the cell. Increased Ca +2 is taken up by mitochondria, which release Reactive Oxygen Species (ROS) and contribute to oxidative stress. BI 749327 can block TRPC6 channel activity and suppress oxidative stress. Meanwhile, hyper-activation of β2AR leads to endosomal β2AR loss, followed by endosomal loss of CFTR (-). Loss of CFTR results in (i) activation of TRADD and downstream activation of IKKαβ to drive NFκB; and (ii) further activation of CFTR-bound TRPC6. NFκB enters the nucleus and drives expression of proinflammatory cytokines IL8 and IL6. Both TRADD and PI3K pathways may be required for optimal NFκB activation, because LY294002 can block inflammation.

Article Snippet: All antibodies were purchased commercially. β-actin (Sigma, A5441); CFTR (R&D Systems, MAB1660); CFTR (SCBT, sc-376683); β2AR (Novus Biologicals, NBP261710); TRADD (SCBT, sc-46653); TNFR1 (Cell Signaling, 3736), IKKβ (SCBT, sc-8014); IκBα (Cell Signaling, 9242); phospho-IκBα Ser32/36 (Cell Signaling, 9246); phospho-NFκB p65 Ser276 (Cell Signaling, 3037); NFκB p65 (Cell Signaling, 8242); phospho-IKKα/β Ser176/180 (Cell Signaling, 2697); IKKα (Cell Signaling, 11930); AKT (Cell Signaling, 9272); phosphor-AKT Ser473 (Cell Signaling, 9271); TRPC6 (Protein Tech, 18236-1-AP); and Ubiquitin (Protein Tech, 80992-1-RR).

Techniques: Blocking Assay, Activity Assay, Activation Assay, Expressing