Sa00001 2

Treated cells were lysed using a lysis buffer (Biosharp) and centrifuged (4°C, 12000 × g, 20 min). The supernatant was collected and the protein concentration was calculated using a protein quantification kit (Biosharp). The protein samples were subsequently boiled (95°C, 5 min) and then cooled down. A sample containing 60 µg of protein was added to each well for electrophoresis (80 V, 30 min; 120 V, 2 h). Subsequently, proteins were transferred onto a membrane (Millipore, MA, United States; 200 mA). The membranes were blocked in 5% non-fat milk (Biosharp) for 2.5 h, washed three times with TPBS (PBS contains 0.1% Tween 20), and incubated with primary antibodies (Ki67: Proteintech, #27309-1-AP, Wuhan, China; Bcl-2:CST, Boston, MA, United States, #15071T; Bax:CST, #5023T; p62:CST, #8025T; LC3-2:CST, #4108S; Beclin1:CST, #3495T; PI3K: CST, #4249T; AKT: CST, #4691T; mTOR: CST, #2983T; p-PI3K: CST, #17366S; p-AKT: CST, #4060S; p-mTOR: CST, #5536S) overnight at 4°C. The membranes were then washed and co-incubated with the corresponding secondary antibodies (Proteintech, #SA00001-1, SA00001-2, Wuhan, China) for 2 h at 20°C, and the protein bands were exposed using the ECL developing fluid (Millipore).

Trypsin digestion was carried out to isolate the cultured cells or cut tissues, after which enhanced radioimmunoprecipitation assay lysis containing protease inhibitor (Boster, Wuhan, China) was added for ultrasonic lysing. Subsequently, bicinchoninic acid protein quantitative kits (Boster) were adopted to determine protein concentration. Next, the proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. After blocking, diluted primary antibodies against AQP4 (1:1000, #59678, CST), HIF1A (1:1000, #36169, CST), MTA1 (1:500, ab71153, Abcam), and GAPDH (1:1000, #5174, CST) were subsequently used to probe the membrane at 4 °C overnight. The following day, the membrane was re-probed with the HRP-labeled secondary antibody (goat anti-rabbit proteintech; SA00001-1; goat anti-mouse proteintech; SA00001-2) for 1 h. Enhanced chemiluminescence working solution (Millipore Corp., Billerica, MA) was added for culture for 1 min. Later, the membrane was placed in the Bio-rad automatic imager for exposure. Image Lab software was applied to quantify the gray levels.