Ab28364

Heart tissue or cells were lysed with radioimmunoprecipitation (RIPA) lysis buffer. The prepared protein sample was separated through 10% SDS‐PAGE and then transferred to PVDF membrane (Millipore). 5% fat‐free milk was used for 1‐hour blocking at room temperature. The membrane was subsequently incubated with primary antibodies overnight at 4°C. The following day secondary antibodies were used for 1‐hour incubation at room temperature before the enhanced chemiluminescence (Millipore) with Amersham Imager 680 (General Electric Company). Primary antibodies included those against CD31 (Abcam, ab28364; Novus, NB100‐2284), VE‐cadherin (Abcam, ab231227; ab33168), α‐SMA (Abcam, ab5694), Vimentin (CST, #5741), collagen I (Abcam, ab34710), collagen III (Abcam, ab7778), Phospho‐AMPKα (Thr172) (CST, #2535), AMPKα (CST, #5831), Smad4 (CST, #38454), TGF‐β (Santa Cruz, sc130348), NOX4 (Proteintech, 14347‐1‐AP) and GAPDH (Proteintech, 10494‐1‐AP).

Serial sections (4 μm) of paraffin-embedded tumor slices were stained for H&E and immunostained using a Dako autostainer for AceCS1 (ACSS2) (Cell Signaling Technology, 3658S), CD31 (Abcam, ab28364), and CAIX (Novus Biologicals, NB100-417). Slides were analyzed using HALO v2.0 software (Indica Labs). Sections stained for CAIX and ACSS2 were analyzed using Cytonuclear v1.5. Briefly, first hematoxylin staining was set as the nuclear stain and ACSS2 staining as stain 1, and we manually checked for stain separation. ACSS2 staining was specified to localize to the nucleus to get the nuclear staining intensities, and individual cells were assigned to have weak, moderate, or strong nuclear intensities in a given area based on pre-defined threshold intensities. This was done for regions of interest (ROIs) that were manually selected in the CAIX image to represent hypoxic or normoxic areas.