Fig. 3

S727-STAT3 inhibition causes mitochondrial dysfunction. a Expression plasmids for wildtype (WT), but not functionally dead S727A mutant, STAT3 increased bioenergetics function in bEnd5 cells rendered STAT3−/− by a CRISPR-Cas9 method. N = 3. b Deletion of STAT3 protein was confirmed by western blot most clearly in cells transfected with the G1A guide RNA (G1A and G1B were replicate conditions of the same Cas9-guideRNA plasmid) which was used for the bioenergetics experiment in (A). c DNA sequence data confirmed a point mutation and frame shift in exon 1. d Pharmacological inhibition of STAT3 with stattic for 2 h in bEnd5 cells reduced mitochondrial bioenergetic measures. n = 3. e pS727-STAT3 is predominantly present in the mitochondria and nucleus as shown in Western blots of protein extracts from sub-cellular fractions. Markers = H3 protein, nucleus (N), α-tubulin, cytoplasm (C), PDH, mitochondria (M). W = whole cell lysate. tSTAT3 = total STAT3. f Cells treated for 4 h with the STAT3 inhibitor, stattic (10 μM), had less pS727-STAT3 in their isolated mitochondria. The blot is representative of three experiments. g pS727-STAT3 was increased in isolated bEnd5 mitochondria treated with okadaic acid (OA: 1 μM) in respiration buffer, as detected by western blotting (3 independent experiments are shown). Stattic did not have any effect. A higher molecular weight band appeared after OA treatment in the pS727-STAT3 blot. A faint band could be seen in the same position after longer exposure of the total STAT3 blot (not shown)