Fig. 3.

Low glucose and metformin-induced mitochondrial damage is associated with the ASK1/Noxa pathway. a Western blot assay of the expression of Noxa on the mitochondria, and total Noxa or Bcl-2 protein in SKOV3 cells treated with 25 mM or 2.5 mM glucose and 8 mM metformin for 24 h. b Quantitation of the ratio of Noxa/Tom20, Noxa/β-actin and Bcl-2/β-actin protein. Data are presented as mean ± SD (n = 3; **P < 0.01). c Western blot assay of the expression of ASK1, p-ASK1, JNK, and p-JNK proteins in SKOV3 cells treated with 25 mM or 2.5 mM glucose and metformin for 24 h. d Quantitation of the ratio of p-ASK1/ASK1 and p-JNK/JNK protein. Data are presented as mean ± SD (n = 3; **P < 0.01). (e and f) After pretreatment of SKOV3 cells with 10 μM NQDI-1, the expression of Noxa protein in the mitochondrial fraction and total Noxa or Bcl-2 protein was measured via western blot analysis (Mit. fraction) following treatment with low glucose and metformin for 24 h. The purity of the mitochondrial fraction was verified by the presence of the mitochondrial protein, Tom20. Quantitation of the ratio of Noxa and Bcl-2 protein. Data are presented as mean ± SD (n = 3; **P < 0.01). g Colocalization of Noxa and Mito-Tracker in SKOV3 cells as observed by confocal microscopy (bar, 10 μm). h-j ASK1 was knocked down in SKOV3 cells using small interfering RNA (siRNA). h Western blot analysis of ASK1 expression. i The effect of ASK1 on mitochondrial membrane potential in SKOV3 and OVCRA3 cells according to 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 the combination of 2.5 mM glucose and metformin). j The effect of ASK1 on cell apoptosis in SKOV3 and OVCRA3 cells as analyzed by flow cytometry. 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)