Phospho akt

Human Bax, Bcl-2 and cleaved caspase-3 antibodies were from Cell Signaling Technology (USA). Cyclin D1, Cyclin A, Cyclin B1, CDK1, phospho-p65, phospho-ERK, phospho-AKT, MMP-2, MMP-7, and MMP-9 antibodies were purchased from Proteintech (USA). GAPDH antibody and HRP-conjugated secondary antibodies were purchased from BIOSS (Beijing, China). Ophiorrhiza pumila was purchased from Foshan Renhui Pharmaceutical Technology Co. (Foshan City, Guangdong Province, China).

For protein samples, we used RIPA (Solaribio, China) to extract total protein from cells and tissue samples mentioned above on the ice. Then equal amount of proteins in each sample was separated by 10–12% SDS-PAGE (Servicebio, China) and transferred onto polyvinylidene fluoride membranes (Millipore, United States). The membrane was treated with a blocking buffer, which consisted of TBST mixed with 5% skim milk, and this process was conducted at room temperature for 1 h. We used TIGD1 (1:1000, 13833-1-AP, proteintech, China), AKT (1:1000, 10176-2-AP, proteintech, China), Phospho-AKT (1:1000, 28731-1-AP, proteintech, China), PI3K (1:1000, 20584-1-AP, proteintech, China), Phospho-PI3K (1:1000, 17366, Cell Signaling Technology, United States), GAPDH (1:100000, 60004-1-Ig, proteintech, China), Beta Actin (1:20000, 66009-1-Ig, proteintech, China) antibodies for incubation at 4°C overnight. Following three washes with TBST, the membrane was subjected to incubation with secondary antibodies. Subsequently, it was treated with enhanced chemiluminescence (ECL) (Easysee Western Blot Kit, China), and digital blot images were captured using an imaging system (Bio-Rad, United States).

C49 (>99% purity) was synthesized in the School of Pharmacy of East China University of Science and Technology. DOX, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Hoechst 33,258 were purchased from Sigma Chemical Co. The primary antibodies, P-gp, Caspase-3, Caspase-9, Caspase-10, Bcl-2, Bcl-xL, p53, phospho-p53, PI3K, phospho-PI3K, Akt, phospho-Akt, β-actin, and Ki-67, were brought from Proteintech.

Transforming growth factor-β (TGF-β, 763104) was purchased from Biolegend (San Diego, CA, USA). Isoprenaline (ISO, I5627) was obtained from Sigma-Aldrich and prepared in DD water. The SMOC2 (SC-376104, 1:500 for western blot and 1:100 for satining) and ILK (SC-20019, 1:500) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against collagen type I (Col I, 1:1000), collagen type III (Col III, 1:1000), phospho-JNK (Tyr185,1:1000), JNK (1:2000), phospho-p38 MAPK (Thr180/Tyr182,1:1000), t-p38 (1:1000), phospho-AKT (1:5000), t-AKT (1:5000) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH,1:4000) were obtained from Proteintech (Wuhan, China). Anti-α smooth muscle actin (a-SMA, 1:100 for staining) antibody was purchased from Abcam (Cambridge, UK).

Cells were extracted by using RIPA lysis buffer containing protease inhibitors (R0010, Solarbio, China), and equal amounts of proteins were separated, and then transferred onto PVDF (0.45 μm) membranes (IPVH00010, Immobilon-P, Millipore). After being blocked with non-fat milk, the membranes were probed with the primary antibodies (1:1000) at 4 °C overnight. The next day, the membrane was washed and incubated with secondary antibodies (SA00001-1 or SA00001-2, Proteintech) for 2 h at room temperature, followed by detection with the chemiluminescence Tanon-5200 (Tanon, Shanghai, China). Antibodies were listed as follows: METTL3 (ab195352, Abcam, Cambridge, UK); phospho-PI3K P85 (TA3242, Abmart, Shanghai, China); PI3K (T40115, Abmart, Shanghai, China); mTOR (T55306S, Abmart, Shanghai, China); Histone H3 (4620S, CST, USA); p-mTOR(5536T, CST, USA); Histone H3 acetyl K9 (ab10812, Abcam, Cambridge, UK); H3K4me3 (PTM-5019, PTM Bio Inc., Hangzhou, China); H3K36me3 (PTM-625RM, PTM Bio Inc., Hangzhou, China); H3K27me3 (ABE44, Merck Millipore, USA). Other antibodies (GAPDH, VGF, phospho-AKT, AKT, KI67) were obtained from Proteintech Biotechnology (Wuhan, China). The original pictures of western blots are listed in Additional file 3.

The protein concentration of the supernatant was measured using a BCA protein assay kit. Next, the proteins were boiled at 95 °C for 10 min and stored at − 20 °C after being converted into equal quality and volume. SDS-PAGE (10%) was used for electrophoresis, and the proteins were then transferred onto PVDF membranes in an ice bath at 350 mA for 60 min. Membranes were blocked with 5% skim milk at room temperature for 1 h and incubated with primary and secondary antibodies. Phosphatidylinositol 3-kinase (PI3K, 20,584–1-AP, 1:1000), protein kinase B (Akt, 60,203–2-Ig, 1:1000), phospho-Akt (66,444–1-Ig, 1:1000), Interleukin-1β (IL-1β, 16,806–1-AP, 1:1000), IL-10 (60,269–1-Ig, 1:1000), and tumour necrosis factor-α (TNF-α, 17,590–1-AP, 1:1000) antibodies were purchased from Proteintech (Wuhan, China). The antibody phospho-PI3K (AP0854, 1:1000) was obtained from Abclonal (Wuhan, Hubei, China), and anti-mouse IgG (7076, 1:3000), anti-rabbit IgG (7074, 1:3000), and β-actin (4970, 1:1000) were obtained from Cell Signalling Technology, Inc. (Beverly, MA, USA). Finally, the protein bands were visualised using Image Lab software and analysed using ImageJ software.

For Western blot analysis, cell lysate was added after the cells were washed three times by PBS. The isolated proteins were quantified and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%), transferred onto a PVDF membrane, and blocked with 5% BSA. After blocking, the proteins were incubated with antibodies overnight at 4°C with primary antibody β-actin (1:3,000, Affinity, USA), Phospho-AKT (1:1,000, Proteintech, USA), AKT (1:1,000, CST, USA), JAK2 (1:1,000, CST, USA), Phospho-JAK2 (1:1,000, CST, USA), p44/42 MAPK (ERK1/2) (1:1,000, CST, USA), Phospho-p44/42 MAPK (ERK1/2) (1:1,000, CST, USA), STAT3 (1:1,000, CST, USA), Phospho-STAT3 (1:1,000, CST, USA), NF-κB (1:1,000, CST, USA), Phospho-NF-κB (1:1,000, CST, USA), PI3K (1:1,000, CST, USA), Phospho-PI3K (1:1,000, CST, USA), and TNF-α (1:1,000, CST, USA), followed by horseradish peroxidase (HRP)-linked anti-rabbit (1:2,000, CST, USA) or anti-mouse (1:2,000, CST, USA). The protein bands were analyzed by Tanon 4600SF (Tiangong Technology Co., Ltd., Shanghai, China) with chemiluminescence substrate.