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. 2015 Jul 1;16(8):1037–1050. doi: 10.15252/embr.201540159

Figure 4.

Figure 4

Stage-specific requirement of Brg1 or Brm for the activation of the differentiation program in C2C12 myoblasts

  1. Schematic representation of the experimental setting, with siRNA delivered to C2C12 cells in GM and EdU pulses in GM or DM 6 h before collecting cells. Cyclin D1 (or control Scr) was downregulated by siRNA in C2C12 cells, which were subsequently interfered for Brm, Brg1, or scrambled sequences (siScr) by small interfering RNA (siRNA). Cells were then cultured in DM for 48 h.
  2. Immunofluorescence analysis of EdU incorporation, myogenin and MyHC in C2C12 collected from experimental conditions indicated in (A). Percentages of positive nuclei or cells are indicated in the top right corner of each panel. Nuclei are counterstained with DAPI. The effect of siBrg1, siBrm, or siScr on EdU incorporation, myogenin and MyHC expression was evaluated in siScr (left panels) or siCyclinD1 (right panels) C2C12 cells. Scale bar, 50 μm.
  3. Relative expression levels of Ccnd1 transcripts were monitored by qRT–PCR in siScr, siBrg1, and siBrm C2C12 cells in GM and DM (48 h). Data are presented as average ± SEM (n = 3).
  4. Quantification of EdU incorporation in nuclei, as percentage of EdU-positive nuclei/total nuclei in randomly selected fields, in siScr, siBrg1, and siBrm C2C12 cells in GM and DM (48 h), in the presence or absence of siCyclinD1. Data are presented as average ± SEM (n > 3). ***P < 0.01 (unpaired Student’s t-test).
  5. Fusion index was calculated by immunofluorescence staining, as percentage of nuclei within MyHC-expressing myotubes, performed in siScr, siBrg1, and siBrm C2C12 cells cultured in DM (48 h), in the presence or absence of siCyclinD1. Error bars represent average ± SEM (n = 3).