Figure 4

JAK-STAT is activated by H₂O₂ and is required for H₂O₂-induced oxidative stress. (A) H₂O₂ activates the JAK-STAT pathway by inducing the phosphorylation of JAK2 and STAT3. C2C12 myotubes were treated with H₂O₂ (2 mmol/L) for 2 h and total cell lysates were subjected to Western blot analysis. (B,C) A pan-JAK inhibitor (JAK I) blocks the H₂O₂-induced phosphorylation (B) and nuclear enrichment (C) of STAT3. (B) C2C12 myotubes were treated with H₂O₂ (2 mmol/L) in the presence or absence of JAK I for 2 h, and total cell lysates were subjected to Western blot analysis. (C) Immunostaining was performed on cultured C2C12 cells. C2C12 myotubes were treated with H₂O₂ (2 mmol/L) for 2 h in the presence or absence of JAK I. STAT3 (red) was stained with STAT3 antibody and nuclei were stained by DAPI (blue), green arrows show the location of nuclei. (D) H₂O₂-induced protein posttranslational modifications are reduced by JAK inhibitors. C2C12 myotubes were treated with H₂O₂ (200 µmol/L, 18 h) in the presence or absence of the pan-JAK inhibitor (JAK I, 1 µmol/L) or R545 (0.5 µmol/L). Protein oxidation and nitration were examined by antibodies against DNP and nitrotyrosine, and protein ubiquitination was detected by anti-ubiquitin antibody. (E) Quantitation of the modified proteins. The gray density of the indicated areas (*) in (D) were quantitated. *p < 0.05. (F,G) The reduction in mitochondrial membrane potential and ATP production with H₂O₂ treatment is reversed by JAK and STAT inhibitors. C2C12 myotubes were treated with H₂O₂ (200 µmol/L, 18 h) in the presence or absence of pan-JAK inhibitor (JAK I, 0.2, 0.5 µmol/L) and STAT inhibitor (stattic; 0.2, 0.5 µmol/L) (n = 6, *p < 0.05). Con, control; Ub, ubiquitin; Nitro., nitrotyrosine; a.u., arbitrary unit.