Recombinant soluble human tgf β1

Transformed human bronchial epithelial cells, BEAS‐2B cells were purchased from Shanghai Cell Bank (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Harry Biotech) supplemented with 15% foetal bovine serum (FBS) (Gibco, 10,099‐141). Then, cells were seeded on 6‐well culture dishes at 1.5 × 10⁵ cells/mL. Experiments were performed when cells reached 70%‐80% confluence. Recombinant soluble human TGF‐β1 (Pepro Tech, 100‐21) was used to induced apoptosis (30 ng/mL, 72 hours) and EMT (10 ng/mL, 48 hours) in vitro, respectively. All experiments were conducted in triplicates and repeated three times.

Recombinant soluble human TGF-β1 and TWEAK were from Peprotech (Rocky Hill, NJ, USA). Recombinant soluble human TNF-α was obtained from eBioscience (San Diego, CA, USA). Purified anti-α-tublin and anti-human Vimentin (V9) monoclonal antibodies (mAbs) SB431542 and AG1478 were from Sigma Chemicals (St. Louis, MO, USA). Anti-human E-cadherin (HECD-1) was from Takara (Tokyo, Japan). N-cadherin and anti-EGFR mAbs were from BD Biosciences (San Jose, CA, USA). Anti-phospho-EGFR (pY845) mAbs was from abcam (Cambridge, UK). Anti-Smad2/3, anti-phospho-Smad2 (Ser465/467), anti-extracellular signal-regulated kinase (ERK), anti-phospho-ERK (Thr202/Tyr204), anti-p38 MAPK, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-Akt, anti-phospho-NF-κB p65 (Ser536) polyclonal antibodies, and anti-ZO-1, anti- Jun N-terminal kinase (JNK), anti-phospho-JNK (Thr183/Tyr185), anti-phospho-Akt (Ser473), and anti-NF-κB mAbs were obtained from Cell Signaling Technology (Beverly, MA, USA). SB202190, SP600125, LY294002, and BAY11-7082 were from Wako Chemicals (Osaka, Japan). AZD6244 was from Selleckchem (Houston, TX, USA). Bronchial epithelial growth medium (BEGM) was purchased from Cambrex (East Rutherford, NJ, USA).