Fig. 4.

Low glucose and metformin-induced activation of ASK1 is associated with ROS. a Flow cytometric analysis of relative ROS level using DCFH-DA in SKOV3 and OVCRA3 cells treated with 25 mM or 2.5 mM glucose and metformin for 24 h. Data are presented as mean ± SD (n = 3; **P < 0.01). b Western blot analysis of the expression of ASK1 and p-ASK1 in SKOV3 cells following treatment with 25 mM or 2.5 mM glucose and metformin for 24 h. Quantitation of p-ASK1/ASK1 protein. Data are presented as mean ± SD (n = 3; *P < 0.05 versus 2.5 mM glucose alone; ^#P < 0.05 versus combination of 2.5 mM glucose and metformin). c After SKOV3 and OVCRA3 cells were pretreated with 40 μM NAC, mitochondrial membrane potential was determined using JC-1 staining and flow cytometric analysis. Data are presented as mean ± SD (n = 3; *P < 0.05 and **P < 0.01 versus 2.5 mM glucose alone; ^##P < 0.01 versus combination of 2.5 mM glucose and metformin). d After cells were pretreated with 40 μM NAC, the expression of Noxa protein in the mitochondrial fraction (Mit. fraction) of SKOV3 cells was analyzed by western blotting following treatment with 25 mM or 2.5 mM glucose and metformin for 24 h. Quantitation of Noxa protein in the mitochondrial fraction. Data are presented as mean ± SD (n = 3; **P < 0.01 versus 2.5 mM glucose alone; ^##P < 0.01 versus combination of 2.5 mM glucose and metformin). e The caspase-3 activity in SKOV3 and OVCRA3 cells was measured by a colorimetric assay kit. Data are presented as mean ± SD (n = 3; **P < 0.01 versus 2.5 mM glucose alone; ^##P < 0.01 versus combination of 2.5 mM glucose and metformin)