Journal: Alcohol, clinical & experimental research

Article Title: Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction

doi: 10.1111/acer.15174

Figure Lengend Snippet: Ethanol-exposed lung fibroblasts induce barrier dysfunction in naïve airway epithelial cells via a TGFβ-dependent mechanism. (A) Human airway epithelial cells (HAEC) were seeded in Transwell inserts and co-cultured with HLF, HELF ± ALK5 inhibitor (SB 431542, 4 μ M), or HELF ± TGFβ1 neutralizing antibody (2 μ g/mL). Both TGFβ1 neutralizing antibody and ALK5 inhibitor were added to the co-cultures in the last 24 h. Transepithelial Electrical Resistance (TER) was measured using EVOM2 after 3 days of co-cultures ( n = 3 biological replicates). (B) RAEC were co-cultured with RLF or RELF for 3 days. An ALK5 inhibitor (SB431542, 4 μ M) was added to co-cultures in the last 24 h ( n = 4–7 biological replicates). Absolute TER ranges are reported in parenthesis below the graphs. *Represents a significant difference compared to AEC co-cultured with LF ( p < 0.05). **Represents a significant change compared to AEC co-cultured with ELF ( p < 0.05).

Article Snippet: Primary rat lung fibroblasts (RLF) were isolated from F344/NHsd rat lungs (Envigo) as previously described ( ) and maintained in complete fibroblast culture media (DMEM 4.5 g/L glucose supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin) at 37°C, 5% CO 2 .

Techniques: Cell Culture

Journal: Alcohol, clinical & experimental research

Article Title: Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction

doi: 10.1111/acer.15174

Figure Lengend Snippet: Co-culture with ethanol-exposed lung fibroblasts altered ZO-1 expression in airway epithelial cells via TGFβ1 signaling. The co-culture of RLF, or RELF, and RAEC was set up as shown in . An ALK5 inhibitor (SB431542, 4 μ M) was added to co-cultures in the last 24 h ( n = 4–7 biological replicates). After 3 days of co-culture, RAEC were collected for total RNA extraction, and RT-qPCR was performed to analyze selected junction proteins gene expression: (A) ZO-1, (B) claudin-5, (C) claudin-18, and (D) occludin. * p < 0.05 compared to RAEC co-cultured with RLF. ** p < 0.05 change compared to RAEC co-cultured with RELF.

Article Snippet: Primary rat lung fibroblasts (RLF) were isolated from F344/NHsd rat lungs (Envigo) as previously described ( ) and maintained in complete fibroblast culture media (DMEM 4.5 g/L glucose supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin) at 37°C, 5% CO 2 .

Techniques: Co-Culture Assay, Expressing, RNA Extraction, Quantitative RT-PCR, Gene Expression, Cell Culture

Journal: Alcohol, clinical & experimental research

Article Title: Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction

doi: 10.1111/acer.15174

Figure Lengend Snippet: Ethanol-exposed lung fibroblasts attenuated overall ZO-1 protein expression and localization in RAEC via TGFβ1 signaling. The co-culture of RLF, or RELF, and RAEC was set up as shown in . An ALK5 inhibitor (SB431542, 4 μ M) was added to co-cultures in the last 24 h ( n = 4–7 biological replicates). After 3 days of co-culture, RAEC were collected for (A) protein expression analyses by Western immunoblot ( n = 3) or (B) immunofluorescence microscopy analysis of ZO-1 (five images per treatment, total >400 cells). Shown are representative immunofluorescent images of RAEC cultured with (C) RLF, (D) RELF, or (E) RELF + SB431542. * p < 0.05 compared to co-cultured with RLF. ** p < 0.05 change compared to RAEC co-cultured with RELF.

Article Snippet: Primary rat lung fibroblasts (RLF) were isolated from F344/NHsd rat lungs (Envigo) as previously described ( ) and maintained in complete fibroblast culture media (DMEM 4.5 g/L glucose supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin) at 37°C, 5% CO 2 .

Techniques: Expressing, Co-Culture Assay, Western Blot, Immunofluorescence, Microscopy, Cell Culture

Journal: Alcohol, clinical & experimental research

Article Title: Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction

doi: 10.1111/acer.15174

Figure Lengend Snippet: Ethanol-exposed lung fibroblasts promoted collagen type 1 and α-smooth muscle protein expression in naïve airway epithelial cells, which was normalized by TGFβ1 signaling inhibitor. The co-culture of RLF, or RELF, and RAEC was set up as shown in . An ALK5 inhibitor (SB431542, 4 μ M) was added to co-cultures in the last 24 h ( n = 3–7 biological replicates). After 3 days of co-culture, RAEC were collected for analysis of mesenchymal proteins by Western immunoblot analyses of (A) collagen type 1A1 (Col1A1) and (B) α-smooth muscle Actin (α-SMA). Selected blot images are depicted above the graphs. * p < 0.05 compared to co-cultured with RLF. ** p < 0.05 change compared to RAEC co-cultured with RELF.

Article Snippet: Primary rat lung fibroblasts (RLF) were isolated from F344/NHsd rat lungs (Envigo) as previously described ( ) and maintained in complete fibroblast culture media (DMEM 4.5 g/L glucose supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin) at 37°C, 5% CO 2 .

Techniques: Expressing, Co-Culture Assay, Western Blot, Cell Culture

Journal: Alcohol, clinical & experimental research

Article Title: Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction

doi: 10.1111/acer.15174

Figure Lengend Snippet: Ethanol-exposed lung fibroblasts induced TGFβ1 and suppressed GM-CSF. The co-culture of RLF, or RELF, and RAEC was set up as shown in . An ALK5 inhibitor (SB431542, 4 μ M) was added to co-cultures in the last 24 h ( n = 4–5 biological replicates). The media were collected on day 3 of co-cultures, and (A) TGFβ1 and (B) GM-CSF levels were measured by ELISA. (C) GM-CSF to TGFβ1 ratios were calculated using GM-CSF to TGFβ1 levels from corresponding samples; the data are presented as a fold change compared to RAEC + RLF group. * p < 0.05 compared to co-cultured with RLF. ** p < 0.05 change compared to RAEC co-cultured with RELF.

Article Snippet: Primary rat lung fibroblasts (RLF) were isolated from F344/NHsd rat lungs (Envigo) as previously described ( ) and maintained in complete fibroblast culture media (DMEM 4.5 g/L glucose supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin) at 37°C, 5% CO 2 .

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture