Jf0582

BEAS-2B cells were plated on cell climbing slices in a 24-well plate. Following the designated treatments, cell climbing slices were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and then incubated with 0.5% triton X-100 for permeabilization. Then, the cells were blocked with 1% BSA for 1 h at room temperature. The primary anti-periostin antibody (1:200, ab152099, Abcam, USA), anti-fibronectin antibody (1:200, JF0582, Huabio, China), and anti-COL1A1 antibody (1:200, BA0325, Boster, China) were used to incubate the cell climbing slices overnight at 4°C. The primary antibody was then removed and fresh PBS was added. After washing with PBS three times, climbing slices were incubated with the secondary antibody goat anti-rabbit IgG (1:500, Abbkine, Hubei, China) and protected from light for 1 h. The nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (1:500) for 5 min. After the final washing steps in PBS (3×10 min), the images were captured under an upright fluorescence microscope (Leica, Berlin, Germany).

BEAS-2B cells were plated on cell climbing slices in a 24-well plate. Following the designated treatments, cell climbing slices were washed with PBS, fixed with 4% paraformaldehyde, and then incubated with 0.5% triton X-100 for permeabilization. Then, the cells were blocked with 1% BSA in PBS for 1 hour at room temperature. The primary anti-TNFSF15 antibody (1:200, DF3053; Affinity, Jiangsu, China), anti-fibronectin antibody (1:200, JF0582; HUABIO), and anti-COL1A1 antibody (1:200, BA0325; Boster) were used to incubate the cell climbing slices overnight at 4°C. The primary antibody was then removed, and fresh PBS was added. After washing with PBS 3 times, climbing slices were incubated with the secondary antibody goat anti-rabbit IgG (1:500; Abbkine Scientific Co., Ltd.) and protected from light for 1 hour. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:500) for 5 minutes. After the final washing in PBS (3 × 10 minutes), the images were captured under an upright fluorescence microscope (Leica, Berlin, Germany).

Total cellular proteins were extracted using RIPA and then subjected to western blot experiments for quantification. To carry out western blot experiments, total proteins were segregated using sodium-dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After brief washing, Tris-buffered saline with 1% Tween 20 (TBST) containing 5% bovine serum albumin (BSA) was used for membrane blocking for 1 hour. Then, the membranes were incubated overnight at 4°C with the primary antibodies including anti-TL1A (1:1,000, ab85566; Abcam, Santa Cruz, CA, USA), anti-DR3/LARD antibody (1:1,000, ab189093; Abcam), Anti-COL1A1 antibody (1:1,000, BA0325; Boster, Wuhan, China), anti-fibronectin (1:1,000, JF0582; HUABIO, Hangzhou, China), anti-alpha smooth muscle actin (1:1,000, SY02-64; HUABIO), anti-E-cadherin (1:1,000, PB9561; Boster), anti-E-cadherin (1:1,000, ER0503; HUABIO), and anti-GAPDH (1:1,000, BA2913; Boster). The following day, the membranes were removed with tweezers and then washed in TBST 3 times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (1:3,000, GB23303; Servicebio, Wuhan, China) at room temperature for 1 hour. After washing with TBST again, the membranes were subjected to chemiluminescence by using enhanced chemiluminescence substrate.