Fig. 7
Melatonin inhibited IP3R1-mediated MAMs via ERK1/2 signaling pathway. A, B Several specific pharmacological inhibitors, including Bisindolylmaleimide XI hydrochloride (Bis XI, a PKC inhibitor), Dorsomorphin (DSM, an AMPK inhibitor), PD98059 (a MEK/ERK inhibitor), Luzindole (a melatonin receptor inhibitor), Ruxolitinib (Ruxo, a JAK inhibitor), EX-527 (a SIRT1 inhibitor) and Wortmannin (a PI3K/Akt inhibitor) were pre-administered to the hepatocytes in the TS + Mel group and then the expression of IP3R1 was quantified (n = 4). C, E Representative blots and quantitative analysis of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (n = 4). F Cell viability (percentage of SS). G The levels of ALT release (fold difference compared with SS). H Quantitative analysis of IP3R1 mRNA expression determined by real-time PCR. I–L ERK1/2 was knocked down by siRNA, after which the cells were subjected to TS with or without melatonin. Representative blots and quantitative analysis of phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and IP3R1 (n = 4). M Quantitative analysis of IP3R1 mRNA expression determined by real-time PCR. N Cell viability (percentage of TS + Scramble RNAi). O The levels of ALT release (fold difference compared with TS + Scramble RNAi). P Representative confocal images of primary hepatocytes double-stained by mitotracker (red) and ER-tracker (green) at × 600 magnification. Q, R Statistical quantification of the colocalization area between mitochondria and ER. S Representative images and quantitative analysis of MitoSOX-stained mitochondria-derived superoxide production. All of the values are shown as the means ± SEM. n = 6 in each group. *P < 0.05, **P < 0.01 vs SS or TS + Scramble RNAi; ##P < 0.01 vs TS or TS + Mel + Scramble RNAi; &P < 0.05 &&P < 0.01 vs TS + Mel