Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: Fig. 3 Identification of Notch target genes in muscle progenitors in differentiation conditions. a Scatter graph of gene expression levels in differentiating Hu5/KD3 cells with (y-axis) or without DAPT treatment (x-axis). RNA was extracted from the differentiating cells after 4 days DAPT treatment and analyzed by RNA-seq. FPKM: fragments per kilobase of exons per million reads mapped. b Numbers of genes differentially (>2-fold) expressed in differentiating Hu5/KD3 cells with or without DAPT treatment. c Experimental design and qPCR analysis of NOTCH2, NOTCH3, PTGER2, and PTGER4 in Hu5/KD3 cells with or without DAPT treatment. RNA was extracted from the cells at day 0, day 4, and day 7. n = 3 samples/group. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t-test. The effect size of Pearson’s r correlation (r) is shown.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Gene Expression, RNA Sequencing, Two Tailed Test

Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: 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.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Staining, Two Tailed Test, Transfection, Plasmid Preparation

Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: Fig. 5 Effects of prostaglandin E2 (PGE2), EP2 agonist (butaprost), EP2 antagonist (TG6-10-1), and overexpression and knockdown of EP2 on myotube formation of a human myoblast cell line, Hu5/KD3 cells. a Experimental design. Hu5/KD3 cells were seeded onto collagen type I-coated 12-well plates (5 × 105 cells/well) and cultured in 10% FBS/DMEM medium with prostaglandin E2 (0.5–100 ng/ml), butaprost (EP2 agonist) (0.01–5 µg/ml), or PE2 antagonist, TG6-10-1 (0.01–10 µM) for 3 days. b Expressions of EP2 (green) and MyHC (MF20, red) on differentiating Hu/KD3 cells w/o DAPT treatment. Scale bar, 100 μm. c Histogram of EP2 signal of mononuclear cells treated with (green) or without (black) DAPT. Y-axis indicates the frequency. Data are shown as mean ± S.D. and analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test. More than 600 cells in each group were measured (n = 3 samples/group). d Representative MyHC staining of differentiating Hu5/KD3 cells treated with PGE2, butaprost, or TG6-10-1 at indicated concentrations. Scale bar, 100 μm. e Fusion index of Hu5/KD3 cells after treatment with PGE2. f Fusion index of Hu5/KD3 cells after treatment with butaprost. g Fusion index of Hu5/KD3 cells after treatment with TG6-10-1.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Knockdown, Cell Culture, Staining

Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: Fig. 6 Overexpression and knockdown of EP2 in Hu5/KD3 cells. a Experimental design of overexpression of EP2 in Hu5/KD3 cells. b Western blotting for EP2 (overexpression: OE1-3) and GAPDH (loading control) 3 days after transfection of the control vector (CMV-DsRed) or CMV-EP2-ires DsRed vectors. Relative expression levels of EP2 (after normalization to GAPDH) are shown below. c Representative image of myotubes (MF20, red) formed by Hu5/KD3 cells after transfection of the control vector (CMV-DsRed) or CMV-EP2-ires DsRed vectors. Scale bar, 100 µm. d Fusion index of (c). n = 4 samples/group. Dunnett’s multiple comparisons test. e RT-qPCR for EP2 (PTGER2) of nontarget (control) short-hairpin (sh) RNA plasmid-transfected and EP2 shRNA plasmid-transfected Hu5/KD3 cells. Plasmids were introduced into Hu5/KD3 cells by electroporation. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t-test, n = 3 samples/group. f Immunoblot analysis for EP2 and GAPDH in nontarget and shEP2 plasmid-transfected Hu5/ KD3 cells. g EP2 signals were normalized to GAPDH. h Representative immunostaining of myotubes formed by nontarget shRNA plasmid or EP2 shRNA plasmid-transfected Hu5/KD3 cells with anti-myosin heavy chain antibody, MF20 (red) and nuclei (DAPI, blue). Scale bar = 200 μm. i Fusion index of Hu5/KD3 cells four days after transfection of shRNA plasmids. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t-test, n = 4 independent experiments. Each dot is an average of 3 views/sample. Both p-value and the effect sizes of Pearson’s r (r) are shown. j Full, uncropped images of western blots in b and f are shown in Supplementary Fig. 10.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Knockdown, Western Blot, Control, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, shRNA, Electroporation, Two Tailed Test, Immunostaining

Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: Fig. 7 Blockage of EP2 signaling promoted differentiation of hiPSC-derived muscle progenitors. a Experimental design. After 6 weeks myogenic induction, muscle progenitors were FACS-sorted and treated with EP2 agonists or an antagonist. b Representative images of myotubes formed by hiPSC- derived muscle progenitors treated with PGE2 (100 ng/ml), butaprost (1 µg/ml), TG6-10-1 (TG, 10 µM), and DAPT (10 µM). Cells fixed with 4% PFA were stained with MF20 (muscle myosin heavy chain, red) and nuclei (DAPI, blue). Scale bar, 200 µm. c, d Fusion index (%) (c) and cell numbers (/mm2) (d). n = 3 samples/group. Three views/sample were analyzed. Data are shown as mean ± S.D. and compared with controls by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Staining

Journal: Communications biology

Article Title: Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

doi: 10.1038/s42003-020-0904-6

Figure Lengend Snippet: Fig. 8 Signal transduction upstream and downstream of EP2 involved in self-renewal of muscle progenitors. a Experimental design. Hu5/KD3 cells were seeded at 1 × 105 cells/well in 24-well plates, and treated with indicated reagents for 4 days. The cells were then immunostained with MF20 (muscle myosin heavy chain) and DAPI (nuclei). b–h Fusion index of Hu5/KD3 cells after 4 days treatment with indomethacin (b), Cox-1 inhibitor (SC-560), Cox-2 inhibitor (valdecoxib) (c), ONO-AE3-208 (EP4 antagonist) (d), forskolin (e), dbcAMP (f), H-89 (inhibitor of PKA) (g), or ESI-09 (Epac inhibitor) (h) are shown. Means ± SD. Dunnett’s multiple comparisons test. n = 3–4 samples/group. Twenty micromolar of forskolin and 10 μM of ESI-09 had toxic effects on the cells: their morphology was quite different from usual unfused mononuclear myoblasts. i Experimental design. Fifteen minutes after addition of PGE2, butaprost, or CAY10684 with or without DAPT, the concentration of intracellular cAMP was measured by ELISA. Half of the cells were cultured for 4 days and examined by immunocytochemistry (i, j). j Intracellular cAMP levels 15 min after addition of PGE2 (25 ng/ml) with or without DAPT (1 μM), butaprost (500 ng/ml) with or without DAPT (1 μM), or CAY10684 (1 μg/ml, EP4 agonist). Means ± SD, triplicates. Tukey–Kramer test. n = 3 samples/ group. k Half of the cells shown in i were cultured for a further 4 days, fixed, and stained with MF20. Scale bar, 100 µm. l Fusion index of i. Note that CAY10684 upregulated cAMP at a similar level to butaprost, but treated cells fused well. Means ± SD. Tukey–Kramer test, n = 4 samples/group.

Article Snippet: The human EP2 shRNA plasmid (sc-40171, Lot# E0709) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotechnology.

Techniques: Transduction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunocytochemistry, Staining