Proteinase inhibitor cocktail

MTT and dimethyl-sulfoxide (DMSO) were obtained from Solarbio Corporation, China. The mouse TNF-α ELISA Kit was purchased from Neobioscience Co., Ltd., China. Trizol reagent was purchased from Invitrogen Corporation, USA. The Reverse Transcription System (A3500 kit) was purchased from Promega Corporation, USA. The Power SYBR Green PCR Master Mix kit was purchased from ABI Corporation, USA. RIPA Lysis Buffer, Super ECL Plus Detection Reagent, Prestained Protein Marker (10-170KD), proteinase inhibitor cocktail, PhosSTOP phosphatase inhibitor, and the ROS detection kit (product no: C1300) were purchased from Applygen Technologies Co., Ltd. COX-2 (H-62) and iNOS (N-20) were purchased from Santa Corporation, USA. The mouse anti-β-actin and HRP-labeled goat anti-rabbit IgG antibodies were purchased from Wuhan Boster Biological Technology, Ltd. The p-p38 and p38α/β (H-147) antibodies were purchased from Beijing ZhongShan-Golden Bridge Biological Technology Co., Ltd., China. Minocycline was purchased from Sigma Corporation, USA. QKL was purchased from Yabao Beizhongda (Beijing) Pharmaceutical Co., Ltd., China.

For protein extraction, the treated cells were harvested and lysed with RIPA lysis buffer (Servicebio, Wuhan, China) containing proteinase inhibitor cocktail (APPLYGEN, Beijing, China) on ice for 15 min, and the supernatants were stored at -80℃ after the centrifugation of lysis liquid. The concentrations of total proteins were measured by a BCA protein assay (Pierce, ThermoFisher Scientific), and equal amounts of proteins were used for western blot to quantify target proteins.
Within western blot, proteins were separated by SDS-PAGE gel electrophoresis and transferred onto PVDF membranes (Millipore). Following blocked with 5% BSA solution for 1 hour, the membranes were incubated at 4 °C overnight with primary antibodies to EMT-related proteins (ZO-1(CST 8193), N-Cadherin(CST 13116), E-Cadherin(CST 3195), β-Catenin(CST 8480), Vimentin(CST 5741)) and GAPDH(CST 5174). Then membranes were incubated with HRP-conjugated anti-rabbit secondary antibody for 2 hours at room temperature, and the protein bands were detected with chemiluminescence detection kit (Immobilon™ Western Chemiluminescent HRP Substrate) and imager.

During protein extraction, cells were collected and lysed by RIPA lysis buffer (Servicebio) with the addition of proteinase inhibitor cocktail (APPLYGEN) on ice for 15 min. The supernatants were stored at −80 °C after centrifugation. The concentrations of total proteins were measured using a BCA protein assay (ThermoFisher Scientific), and 10 μg proteins of each sample were used for western blotting. Proteins were separated by SDS-PAGE gel electrophoresis and transferred onto PVDF membranes (Millipore). Following blocked with 5% BSA solution for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies to CFP1 (1:2000, ab198977), H3K4me3 (1:1000, CST #9751), GAPDH (1:1000, CST#2118), SETD1A (1:5000, Proteintech), SETD1B (1:1000, ab300479), H3K4me1 (1:5000, ab176877), H3K4me2 (1:2000, ab32356), H3K9me3 (1:1000, ab176916), Histone H3 (1:5000, Proteintech), TGFBR3 (1:500, sc-74511), TGFB1 (1:500, sc-130348), SMAD6 (1:500, sc-25321), TCF1/TCF7 (1:1000, CST#2203), LEF1 (1:1000, CST#2230), c-MYC (1:1000, CST#18583), β-Catenin (1:1000, CST#8480), and Tubulin (1:1000, ab7921). Then membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody (1:3000, CST#7074 or CST#7076) for 2 h at room temperature, and the protein bands were detected with the chemiluminescence detection kit (Immobilon™ Western Chemiluminescent HRP Substrate) and imager.