Sb 505124

The following antibodies (Abs) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA): p-SMAD1 (Ser206), p-SMAD2 (Ser465/467), p-SMAD3 (Ser423/425), SMAD2/3, HA-Tag, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-MAPK14 (Thr180/Tyr182), MAPK14, and p-JNK1/2 (Thr183/Tyr185). Abs against GAPDH, RUNX2, COL10A1, MMP13, SMAD1, SMAD4, ITGB1, JNK1/2, THOC1, p-SMAD1 (Ser463/Ser465), and anti-phospho-serine/threonine antibody were from Abcam (Cambridge, UK). Abs against COL2A1, BMPR1A, and BMPR1B were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). p-ITGB1 (Thr788) and Flag-Tag Abs were obtained from Sigma-Aldrich (St. Louis, MO, USA). Goat anti-rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP) secondary antibodies were purchased from Thermo Scientific (Waltham, MA, USA). Goat anti-rabbit IgG (H+L), F(ab′)2 fragment (Alexa Fluor® 555 Conjugate) secondary antibodies were from CST. Anti-human IgG antibody was purchased from Abcam.
Blocking antibodies against human integrin beta1, recombinant human interleukin-1 beta, and recombinant human BMP2 were obtained from R&D systems. Purified COL2A1 was purchased from Chondrex, Inc. (Redmond, WA, USA). U0126 and SP600125 were from CST. SB505124 was obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells were transfected with miR-1208 mimics, miR-mimic-control, circCREBBP siRNAs, TGFβ2 siRNAs, or si-NC (RiboBio, Guangzhou, China) at 50 nM, whereas miR-1208 inhibitor or inhibitor control (RiboBio) was used at a concentration of 100 nM. The overexpression vector pLC5-circCREBBP was constructed using amplified DNA fragments, including the sequence of full-length human circCREBBP_007 cDNA (713 bp, NM_004380) obtained from Geneseed (Guangzhou, China). Human pGenesil-1-si-h-circCREBBP based on valid siRNA-circCREBBP sequences was purchased from HanBio (Shanghai, China). The specific sequences are listed in Supplementary Table 3. Specific inhibitors of ALK5 (SB-505124, HY-13521, 1 μM) and ALK1 (K02288, HY-12278, 0.5 µM) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

The GH3, AtT20, MMQ, and RC-4BC cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). GH3, AtT20, and MMQ cell line Twere cultured in Ham's F-12 K medium (L450KJ, BasalMedia) supplemented with 2.5% FBS (S615JY, BasalMedia), 15% horse serum (26,050,088, ThermoFisher), and 1% penicillin/streptomycin (C100C5, NCM biotech. RC-4BC cells were cultured in DMEM (L130KJ, BasalMedia) with 10% FBS, 5 ng/ml recombinant rat EGF (ab290070, Abcam), and 1% penicillin/streptomycin. The following inhibitor and recombinant protein were used: recombinant INHBA (C687, Novoprotein), SB-505124 (HY-13521, MedChemExpress), A 83–01 (HY-10432, MedChemExpress), and recombinant human Follistatin (10685-H08H, Sino Biological).