Fig. 4

Phosphorylation of GSR by AMPKα1 was responsible for ROS maintenance. a Knockdown efficiency of siRNA targeting AMPKα1 was validated by western blot analysis. b RKO and HCT116 cells were transfecting with siRNA targeting AMPKα1 before incubation with 5 mM glucose or glucose-free medium and the cellular ROS production was detected with DCF-DA staining. c RKO and HCT116 cells expressing control-shRNA (sh-NC), sh-2 RNA, or sh-3 RNA were cultured in glucose-free medium with or without NAC (3 mM) for 18 h before cell death was quantified by trypan exclusion staining. d RKO and HCT116 cells expressing sh-NC, sh-2 RNA, or sh-3 RNA were cultured in glucose-free medium for 12 h before measurement of the intracellular NADP⁺/NADPH ratio. e Total cell lysates from RKO and HCT116 cells cultured with or without glucose were subjected to Phos-tag gel electrophoresis and immunoblotted for GSR. pGSR: red triangle. Top shows immunoblot for GSR without Phos-tag. f Total cell lysates from HCT116 cells treated with Compound C or phenformin were subjected to phos-tag gel electrophoresis and immunoblotted for GSR. *P < 0.05, **P < 0.01, Student’s t-test