Anti hif 1α

We performed immunohistochemical staining and western blotting using methods that have been described previously [24 (link)]. Anti-YAP (1:100, immunohistochemistry), Anti–phosphorylated-YAP (p-YAP) (Ser127) (1:1000, western blot), and anti–p-LATS1 (Ser909) (1:1000, western blot) were from Cell Signaling Technology (Shanghai, China). Anti-YAP (1:1000, western blot), anti-LDHA (1:1000, western blot), anti-PKM2 (1:1000, western blot), anti-Glut1 (1:1000, western blotting), anti-PGK1 (1:1000, western blot), anti–large tumor suppressor kinase 1 (LATS1, 1:1000, western blotting), and anti–HIF-1α (1:1000, western blot) rabbit monoclonal antibodies were from Proteintech (Wuhan, China). Anti–matrix metalloproteinase 2 (MMP2, 1:1000, western blot), anti-MMP9 (1:1000, western blot), horseradish peroxidase–linked secondary antibody (1:5000, western blot), and anti–β-actin (1:3000, western blot) were from Abbkine (Wuhan, China). The protein bands were analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).

Total proteins were extracted from cultured cells by RIPA buffer. The results showed that the BCA detection method (Beyotime, Nantong, Jiangsu, China) quantitatively detected all proteins. The protein samples separated by PAGE (polyacrylamide gel electrophoresis) were transferred to the PVDF membranes (EMD Millipore, Billerica, Massachusetts, USA). The PVDF membranes were blocked in 5% skim milk for 1 h, then incubated with specific antibodies overnight at 4 °C, and finally incubated with the secondary antibodies for 2 h at room temperature. The membranes were visualized using enhanced chemiluminescence solution (Pierce Biotechnology, Inc. USA). The experimental results were analyzed by ImageJ analysis software. The following antibodies were recorded: anti-GAPDH, anti-FLAG-tag, anti-HIF1α, anti-YTHDF2 (Proteintech, Wuhan, Hubei, China), anti-AKT, anti-Phospho-AKT (Ser473), anti-ERK1/2, anti-Phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technologies, Danvers, MA, USA), anti-Phospho-mTOR (Ser2448), anti-mTOR, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-Snail1, anti-METTL3, anti-METTL14.

The cells were collected and RIPA lysis buffer (Meilun Biotechnology Co., Ltd., Suzhou, China) containing PMSF (1 mM) (Meilun Biotechnology Co., Ltd., Suzhou, China) was added for protein extraction. Total protein concentration of samples was detected by BCA protein assay. After diluting the protein sample to the same concentration with RIPA lysis solution, added 5 × loading buffer, boiled in 100 °C water bath for 5 min, and store at -80 °C after cooling on ice. 30 μg total protein samples were taken from each group, separated by SDS-PAGE gels electrophoresis and transferred to PVDF membranes. After sealing with 5% bovine serum albumin (Albumin from bovine serum, BSA), the membranes were incubated with primary antibodies anti-VEGF (1:2000, 66,828–1-IG; proteintech), anti-HIF-1α (1:2000, 66,730–1-IG; proteintech), or anti-GAPDH (1:2000, 60,004–1-IG; proteintech). Following incubation with the secondary antibody solution of HRP Goat Anti Mouse IgG (H + L) (1:5000, proteintech). The PVDF membranes were color-coded by chemiluminescence method and luminescence detection by gel imaging system Versa DocTM Imaging System to collect strip images. GAPHD was used as internal reference to calculate the relative expression levels of each target protein by Image Lab analysis software (National Institutes of Health).

Western blot assay was carried out as previously described [18 (link)]. Total protein was extracted with RIPA lysis buffer (Solarbio, China) supplemented with protease inhibitors (Roche Applied Science, Switzerland). The primary antibodies used were anti-E-cadherin (1 : 500, Santa Cruz, USA), anti-Vimentin (1 : 500, Santa Cruz, USA), anti-Twist (1 : 500, Santa Cruz, USA), anti-HIF-1α (1 : 500, Proteintech, China), anti-Slug (1 : 1000, Cell Signaling Technology, USA), anti-Snail (1 : 1000, Cell Signaling Technology, USA), anti-MMP2 (1 : 1000, Cell Signaling Technology, USA), anti-MMP9 (1 : 1000, Cell Signaling Technology, USA) and anti-GAPDH (1 : 5000, Proteintech, China). Horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1 : 1000, Beyotime, China) was used as secondary antibodies. All experiments were performed in triplicate.

For immunohistochemical staining, after antigen retrieval, the slides were blocked with a 5% BSA solution for 1 ​hour and incubated with mouse anti-CD68 (1:100, Invitrogen, USA), anti-iNOS (1:100, Abcam, UK), anti-CD163 (1:1000, Proteintech, USA), anti-TNFα (1:100, ZenBio, China), anti-IL1β (1:100, Proteintech, USA), anti-IL10 (1:100, Proteintech, USA), anti-TGFβ1 (1:100, Proteintech, USA), anti-MMP9 (1:100, Proteintech, USA), anti-OCN (1:100, Affinity, China), anti-HIF1α (1:100, Proteintech, USA) and anti-SDHB (1:100, Proteintech, USA) at 4 ​°C overnight. Goat anti-rabbit IgG (1:100, Servicebio, China) was used as a secondary antibody and the nuclei were stained using 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Servicebio, China). Images were captured using Aperio AT2 (Leica, German).

Immunohistochemistry and immunofluorescence were performed as previously described^{48 (link)}. Anti-GBE1 (1:300; Abcam), anti-Ki-67 (1:300; Abcam), anti-caspase 3 (1:300; Abcam), and anti-HIF1α (1:300; Proteintech, Rosemont, IL, USA) were used as primary antibodies. For immunofluorescence, Cy3- and FITC-conjugated secondary antibodies (1:500; BioLegend, San Diego, CA, USA) were used to detect primary antibodies. The samples were then imaged using a fluorescence microscope (IX71; Olympus, Japan).