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. 2016 Oct 15;143(20):3674–3685. doi: 10.1242/dev.139360

Fig. 5.

Fig. 5.

JMJD3c facilitates the MYOD1-mediated muscle differentiation of hESCs. (A) Schematic of the differentiation protocol. JMJD3c-hESCs were treated with or without Dox on Days 1 to 2 after plating and were transfected with synthetic mRNAs for MYOD1 or Emerald three times on Days 2 and 3. The cells were collected for each experiment on Day 5. (B) qRT-PCR analyses of the expression of myogenic genes in MYOD1-differentiated cells with (+Dox) or without (−Dox) Dox treatment. −, no transfection; Em, Emerald transfection; MYOD1, MYOD1 transfection. The expression levels were normalized to GAPDH. The error bars indicate the s.e.m. from three independent biological replicates. (C-E) ChIP-qPCR analyses of H3K27me3 (C), H3K4me3 (D) and H3K27ac (E) in the MYOG and MEF2C promoters of MYOD1-transfected cells with (+Dox) or without (−Dox) Dox treatment. Three (a-c) and two (a,b) regions of the MYOG and MEF2C promoters were tested, respectively. In human myoblasts, the MYOG (a,b) and MEF2C (a) regions are only enriched for H3K27ac, but not H3K4me3, whereas the MYOG (c) and MEF2C (b) regions are enriched for both markers (see also Fig. S6). T, positive control; SOX1-3′, negative control. The error bars indicate the s.e.m. from three independent biological replicates. *P<0.05, t-test. (F) Immunostaining for MHC in the cells overexpressing JMJD3c (+Dox), MYOD1 or both JMJD3c and MYOD1 (+Dox +MYOD1). Scale bar: 200 μm. (G) The percentage of nuclei contained within the MHC-stained cells out of total cells. The error bars indicate the s.e.m. from three independent biological replicates. *P<0.01, t-test. (H) MHC immunostaining in the MYOD1-transfected cells overexpressing JMJD3c or the JMJD3c mutant. The percentage of nuclei contained within the MHC-stained cells is shown. n=3. Scale bar: 200 μm. (I) RNA-seq analyses of upregulated genes in the myogenic cells that were induced from hESCs (ES-iMuscle) and human skeletal myotubes (Myotube). Upregulated genes were determined using ExAtlas (fold-change >4 in induced myogenic cells and >10 in myotubes relative to hESCs, z-value >4).