Figure 3

Overexpression of constitutively active STAT3 induces oxidative stress. (A) Constitutively active STAT3 (STAT3C) induces the protein expression of Bim (Bcl2l11), but not the components in electron transportation chain. Twenty microgram of pcDNA3-STAT3C plasmid, together with 2 µg of pcs2GFP plasmid, was electroporated into mouse TA muscle. GFP+ fibers were isolated 10 d later and total protein extracts were subjected to Western blot analysis (n = 3 pairs of mouse muscles). Left pane shows the efficiency of electroporation. Green fibers are those with the GFP expression. Right panel shows the results of Western blot analysis. C1: NDUFB8, C2: DHB, C3: UQCRC2 and C5: ATP5A. (B) Quantitative data of protein expression changes in (A). ImageJ was used for quantitation of gray density on the Western blot (n = 3 pairs, *p < 0.05). (C,D) Constitutively active STAT3 (STAT3C) induces protein oxidation and ubiquitination in vivo. (C) Samples were prepared as in (A), and Western blot analysis was performed. Ponceau S (Pon S) is shown as loading control. (D) Quantitative data of protein expression changes in (C). ImageJ was used for quantitation of gray density on the Western blot (n = 3 pairs, *p < 0.05). (E) Activated JAK2 suppresses mitochondrial membrane potential. Constitutively active JAK2V617F was transfected into HEK293 cells for 3 d, followed by JC-1 assay. JC-1 index is the ratio of aggregated JC-1 over monomeric JC-1 (n = 6, *p < 0.05). (F) Activated STAT3 (STAT3C) alters the expression of genes involved in regulating mitochondrial function. Plasmid expressing constitutively active STAT3C was transfected into HEK293 cells for 3 d. Total RNA was collected and quantitative PCR was performed to examine the mRNA levels (n = 4 experimental/control pairs, *p < 0.05). Con, control; arb. unit, arbitrary unit.