T40115

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.

Fresh tissues or adherent cells were washed twice with PBS and then lysed on ice for 1 h with RIPA lysis buffer (Servicebio, China) containing a cocktail and PMSF. After centrifugation, total protein concentrations were determined using a BCA protein assay kit (Biosharp, China). Individual samples (20 μg/lane) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10–12% gels and transferred onto polyvinylidene difluoride membranes. After incubation with 5% dry milk in TBST at room temperature for 1 h, the membranes were washed and incubated with pan-anti-Kla (PTM-1401, PTM Bio, China), anti-H3K18la (PTM-1406, PTM Bio, China), anti-USP39 (23865-1-AP, Proteintech, China), anti-PGK1 (17811-1-AP, Proteintech, China), anti-PI3K (T40115, Abmart, China), anti-p-PI3K (T40116, Abmart, China), anti-AKT (T55561, Abmart, China), anti-p-AKT (T40067, Abmart, China), anti-HIF-1α (TA1009, Abmart, China), anti-β-Actin (AC038, ABclonal, China), and anti-H3 (17168-1-AP, Proteintech, China) at 4 °C overnight. After washing the membranes, the bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence (ECL) kit (Biosharp, China). The relative expression or modification levels of the targets to those of β-Actin or H3 were determined by densitometric analysis using the ImageJ software.