Fig. 7: Ruxo downregulates DRP1 through inhibition of JAK1/2-STAT3 signaling pathway activation and causes mitochondrial division dysfunction.

A RT-PCR were used to determinate the levels of mitochondrial fission-related genes, after treating with Ruxo (0, and 80 μM) for 24 h. B, C RT-PCR were used to measure the DRP1 RNA at various time intervals (0, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h) with treatment of Ruxo (80 μM). And western blot was used to determinate the protein levels of DRP1, PARP, c-PARP, full-length GSDME and GSDME-N terminus. D, E RNA and protein change of STAT3 and DRP1 were measure after transfection of si-STAT3 by RT-PCR and western blot. F, G Schematic diagram of DRP1 promoter containing two putative STAT3 binding sites. STAT3 transcriptional regulation of DRP1 luciferase reporter gene assay. H After transfected with DRP1 overexpression plasmid for 48 h, confocal laser (Leica STED, Germany) was used to observe mitochondrial morphological changes in 8505C and KHM-5M cells, treated with Ruxo (0, 40 μM for 0 h, 12 h), where mitochondrial were stained by Mito tracker Deep Red FM. I After transfected with DRP1 overexpression plasmid for 48 h, confocal laser was used to observe mitochondrial morphological changes in 8505C and KHM-5M cells, treated with Ruxo (0, 40 μM for 0 h, 12 h), where mitochondrial were stained by HSP60 antibody. J, K Quantification of mitochondrial length of H and I. Data are shown as mean ± SD for n = 3, analyzed by one-way ANOVA using the Holm-Sidak method (B, D, I, J), or two-way ANOVA using the Tukey method (G) for multiple comparisons. *p < 0. 05, **p < 0. 01, ***p < 0. 001, ****p < 0. 0001.