Research Paper Volume 6, Issue 10 pp 820—834

Figure 4. PARP-1 inhibition prevents the H2O2-induced myotube death.

Total RNA and cell lysates were isolated from myotubes either treated or not treated with H₂O₂. (A) Global cellular protein PARylation was determined in the total cell lysate by immunoblots. (B) PARP-1 mRNA and protein levels were determined in total mRNA and cell lysates, respectively. GAPDH was used as a loading control. (C) Myotube survival (%) in the presence or absence of H₂O₂ was determined by MTT assay at the indicated time-points. (D) Global cellular protein PARylation was determined in total cell lysates from either H₂O₂-treated or non-treated myotubes either in the presence or absence of PJ34 by immunoblots. (E) Myotube survival (%) was determined by the MTT assay in myotubes treated with the conditions similar to ‘D’. (F) Myotubes were transfected with non-specific or PARP-1-targeting siRNAs. PARP-1 protein abundance were analyzed 48 h after transfection by immunoblots. (G) Global cellular protein PARylation was determined in total cell lysates from either H₂O₂-treated or non-treated myotubes either in the presence or absence of PARP-1 by immunoblots. (H) Myotube survival (%) was determined by a MTT assay in myotubes treated with the conditions similar to ‘G’. (I) SIRT-1 mRNA (left side) and protein (right side) levels were determined in total mRNA and cell lysates, respectively. (J) PARP-1 acetylation levels were estimated from immunoblots after IP with the conditions similar to ‘G’. (K) PGC-1α acetylation levels were determined in total cell lysates from either myotubes treated with H₂O₂ or non-treated myotubes with or without PJ34 or RSV by immunoblotting after IP. Global cellular protein PARylation (L) or myotube survival (%) was determined in total cell lysates from either myotubes treated with H₂O₂ or non-treated myotubes with or without resveratrol. IP, immunoprecipitation; RSV, resveratrol.