Figure 2
PARP1 re-expression reduces the induction of MyoD target genes during differentiation. (a) Western blot analysis of PARP1 and MyoD levels in shPARP1 cells stably transfected with the PARP1 expression vector (shPARP1-PARP1), compared to shPARP1 control cells transfected with the empty vector (shPARP1-CTRL). Proteins were extracted from cells proliferating in growth medium (GM) or kept for 24 and 48 h in differentiation medium (DM 24 h and DM 48 h respectively). ERK1 was used as an invariant control. One representative experiment of two independent ones is shown. Full length blots are presented in Fig. S9. (b) RT-qPCR analysis of p57, myogenin (MyoG), p21 and Mef2C induction in shPARP1-PARP1 compared to shPARP1-CTRL cells. RNA levels were analysed 24 and 48 h after the shift to differentiation medium. Expression levels, relative to those of Tbp RNA and expressed as fold change respect to the control at 24 h, are reported as the mean ± SEM of three independent experiments. Statistical significance: p-value < 0.05 (*), < 0.01 (**), < 0.001 (***). (c) Quantification of the immunofluorescence analysis of MHC-positive myotubes in CTRL, shPARP1, shPARP1-CTRL and shPARP1-PARP1 cells. The percentage of nuclei in MHC-positive cells was calculated as the ratio of the nuclei contained in MHC-positive cells to total number of nuclei, 72 h after shift to the differentiation medium. Representative images are presented in Fig. S3.
