P70s6k

The compound Her, synthesized by Dr. Zhi-guo Mang, was dissolved in dimethyl sulfoxide (DMSO) as a 50 mM stock solution and stored at −80 °C. Bafilomycin (BafA1, #HY-100558), hydroxychloroquine sulfate (HCQ, #HY-B1370) and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA, #HY-D0940) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Compound C (CC, S7306) was purchased from Selleck Chemicals (Houston, TX, USA). N-acetylcysteine (NAC, #S0077) and the Annexin V-FITC apoptosis detection kit (#C1062L) were purchased from Beyotime Biotechnology (Shanghai, China). Primary antibodies against p-AMPKα (Thr172, 1:1000, #2535) and p-p70S6K (Thr389, 1:1000, #9234) were obtained from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against LC3 (1:1000, #14600-1-AP) and SQSTM1/p62 (1:1000, #18420-1-AP) were obtained from Proteintech Group (Wuhan, Hubei, China). The following antibodies were obtained from Abclonal Technology (Wuhan, China): ATG5 (:1000, #A0203), AMPKα (1:1000, #A12718), p-mTOR (Ser2448, 1:1000, #AP0094), mTOR (1:1000, #A2445), p70S6K (1:1000, #A2190), β-actin (1:5000, #AC038) and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (H + L) (1:5000, #AS014).

CANA, MG132, LW-6 and cycloheximide (CHX) were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Cobaltous chloride (CoCl₂) was obtained from Sigma-Aldrich (Chicago, NJ, USA). The specific primary antibodies used for Western blotting analysis against protein kinase B (AKT) (1:1000), p-AKT (1:1000), mTOR (1:1000), p-mTOR (1:1000), HIF-1α (1:1000), fibronectin (FN-1) (1:1000), lactate dehydrogenase A (LDHA) (1:1000), glucosetransporter1 (GLUT1) (1:1000), hexokinase 2 (HK2) (1:1000), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) (1:1000), β-catenin (1:1000), E-cadherin, snail, zonula occludens-1 (ZO-1) (1:1000) and talin (1:1000) were purchased from Cell Signaling Technology (Boston, MA, USA), while actin (1:50,000) was purchased from Sigma-Aldrich^® (Darmstadt, Germany), and vascular endothelial growth factor A (VEGFA) (1:500), P70S6K (1:2000), and p-P70S6K (1:2000) were purchased from Abclonal (Wuhan, China). The secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).