Gt11 a

Cells were seeded on coverslips and allowed to adhere overnight. Cells were then fixed in 4% paraformaldehyde (PFA) followed by permeabilization with 0.5% Triton X-100. Primary antibodies were diluted in 3% BSA and applied overnight at 4 °C. The following primary antibodies were used: GLUT1 (1:250; Alpha Diagnostic; GT11-A) and p63 (1:200; Biocare Medical; CM163A). Fluorophore-conjugated secondary antibodies were used to visualize primary antibody staining (1:400; Life Technologies; A-11070, T-6390). Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and mounted using Vectashield Mounting Medium (Vector Labs) and viewed under a fluorescent microscope (Nikon Eclipse Ni-U).

Cells were lysed in CST lysis buffer (Cell Signaling Technology) supplemented with MG132 (40 μM; Calbiochem) and cOmplete Protease Inhibitor Cocktail (Roche). Lysates were separated by SDS–PAGE and immunoblotting performed on polyvinylidene difluoride transfer membranes (Fisher Scientific) with primary antibodies diluted in 5% BSA overnight at 4 °C. The following antibodies were used for immunoblotting: GLUT1 (1:1,000; Alpha Diagnostic; GT11-A), p63 (1:1,000; Biocare Medical; CM163A), CK5 (1:1,000; Abcam; ab52635), HIF-1α (1:1,000; BD Transduction Laboratories; 610959), AKT and Ser473-p-AKT (1:1,000; Cell Signaling; 9,272 and 9,271, respectively), β-actin (1:5,000; Sigma; A5441). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) were diluted 1:5,000 in 5% skim milk, followed by detection with SuperSignal West Femto or Pico substrate kits (ThermoFisher). Uncropped images of immunoblots are provided as Supplementary Fig. 22.