Fig. 4: NOTCH3-high muscle progenitors were self-renewing, and highly expressed PTGER2 and PTGER4.

a Experimental design. Hu5/KD3 cells were seeded onto collagen type I-coated 60-mm dishes (25,000 cells/cm²) and cultured in 10% FBS/DMEM for 3 days. Then FACS sorting and qPCR analysis were performed. Sorted cells were replated and cultured for a further 3 days, fixed, and stained with MF20 (muscle myosin heavy chain) and DAPI (nuclei). b FACS plot. Sorted NOTCH3-high (N3+) and NOTCH-negative (N3−) fractions are shown by ellipses. c Representative muscle myosin heavy chain staining of differentiating NOTCH3-negative (N3−) and NOTCH3-positive (N3+) Hu5/KD3 cells. Cells were stained with MF20 (MyHC, red) and nuclei (DAPI, blue). Scale bar = 100 μm. Fusion index, percentage of MYOGENIN-positive cells, and cell numbers are shown as dot plots. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t-test. n = 3 samples/group. Three views/sample were analyzed. Both p value and the effect sizes of Pearson’s r correlation (r) are shown. d qPCR for NOTCH2, NOTCH3, PTGER2, PTGER4, HEY1, and HES1 of NOTCH3-negative (N3−, blue ellipse in b) and NOTCH3-positive (N3+, red ellipse in b) cell fractions. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t-test. n = 3 samples/group. Both p-value and the effect sizes of Pearson’s r (r) are shown. e Hu5/KD3 cells were transfected with hNICD3-copGFP plasmid or a parental (empty) plasmid, and 3 days later, copGFP-positive cells were collected by FACS and re-seeded on collagen-coated dishes. Two days later, total RNA was extracted from the cells and qPCR for PTGER2(EP2) and PTGER4(EP4) was performed. Unpaired two-tailed Student’s t-test, n = 3 samples/group.