Fig 1: Isolation and characterization of iMyoblasts.(A) Schematic of a three-stage transgene-free iPSC induction, iMyoblast reserve cell isolation, and iMyotube differentiation protocol. Images of S2 cells and iMyoblasts immunostained with MYOD1 antibody, and S3 iMyocytes and iMyotubes immunostained with MF20 myosin antibody. Nuclei are stained with DAPI. Scale bars=100 µm. Quantification of % MYOD1+ S2 cells and iMyoblasts, fusion index (the percentage of nuclei within MF20+ cells containing =2 nuclei ) and % nuclei within MF20+ cells for S3 cells and iMyotubes are shown on the right. For quantification, each dot corresponds to the % MYOD1+ cells or fusion index in an individual image. Data are presented as mean ± SEM for each condition. (B) qPCR assays of PAX3, PAX7, and MYOD1 in bMyoblasts (17Ubic) and iMyoblasts (17UM) normalized to RPL13A. (C) qPCR assays normalized to RPL13A of proliferating (top, iMyoblasts) or Day 7 differentiated (bottom, iMyotubes) Ctrl 17UM iMyotubes with increasing passage (P) numbers. (D) Flow cytometry of CD56 and CD82 cell surface markers for bMyoblasts (17Ubic, 17Abic) and iMyoblasts (17UM, 17AM). Table below summarizes flow cytometry assays of iMyoblast surface markers in Ctrl (17UM) and FSHD1 (17AM, 15AM) cell lines. (E) MF20 immunostaining of CD56+/CD82+ or CD56-/CD82+ Ctrl (17UM) iMyotubes after 7 days of differentiation. Scale bars=100 µm. Figure 1—source data 1.Source data for Figure 1.
Fig 2: The regulatory effects of miR-30b-5p and Pax3 on gene expression.(A) Artificial upregulation and downregulation of miR-30b-5p expression using mimics and inhibitors, respectively. Expression of miR-30b-5p, Pax3, and SLC7A11 in cells was assessed using PCR. NC: transfection of negative control of miRNA vectors; OV: miR-30b-5p overexpression; KO: miR-30b-5p knockdown. *p < 0.05, **p < 0.01, compared with the control group. (B and C) Luciferase reporter assay was performed to identify the putative binding sites (seed sequences) for hsa-miR-30b-5p in the 3'-UTR of pax3 and SLC7A11 mRNA, as predicted by the bioinformatic analysis webs, including TargetScan, miRanda, and PicTar. The wild-type (WT) pax3 and SLC7A11 3'-UTR, as well as their mutant versions (MT), with mutations in the putative binding sites, were synthesized and inserted into a pmirGLO vector at XhoI and NotI restriction sites. HTR-8/SVneo cells were transfected with the WT or MT constructs, along with either hsa-miR-10b-3p mimics or negative control. The activity of firefly luciferase was measured and normalized to that of Renilla luciferase. Pax3(W): wild-type pax3 constructs; Pax3(M1) and Pax3(M2): two mutant type of Pax3 constructs; SLC7A11(W) and SLC7A11 (M): wild-type and mutant type of Pax3 constructs, respectively; Mimics: hsa-miR-10b-3p mimics. *p < 0.05, **p < 0.01, compared with the Pax3(W) and SLC7A11(W) groups; #p < 0.05, ##p < 0.01, compared with the Pax3(W) + Mimics and SLC7A11(W) + Mimics groups. (D) ChIP assay was performed to determine whether Pax3 could bind to the promoters of SLC7A11, FPN1 and TFR1 genes. **p < 0.01, ***p < 0.001, compared with the IgG group. (E) Pax3 in HTR-8/SVneo and TEV-1 cells was knocked down, followed by the evaluation of protein levels of SLC7A11, FPN1, and TFR1 using western blot assay. Pax3 KO: Pax3 knockdown. *p < 0.05, **p < 0.01, compared with the control group.
Fig 3: The regulatory effect of the HOXD8/DIAPH2-AS1 network on PAX3 expression(A) DIAPH2-AS1 and RP11-445K13.2 were knocked down in HTR-8/SVneo cells under hypoxia using RNA interference technology. (B) The expression of PAX3 in HTR-8/SVneo cells under hypoxia was measured by Western blot assay after DIAPH2-AS1 and RP11-445K13.2 knockdown. (C) The combination of LSD1 and DNMT1 with histone 3 was analyzed by chromatin immunoprecipitation assay after HOXD8 and DIAPH2-AS1 knockdown. (D) RIP assay was performed on HTR8/SVneo cells to investigate interactions between LSD1 and DIAPH2-AS1. The enrichment of DIAPH2-AS1 on LSD1 was determined by PCR after HOXD8 knockdown or not. (E) MeDIP-PCR assay was performed to evaluate the methylation levels at the promoter region of Pax3 gene after the knockdown of HOXD8 and DIAPH2-AS1. *P< 0.05, and **P< 0.01. Nor: normoxia; LO: Low oxygen (hypoxia); HOX(-): HOXD8 knockdown; DIA(-):DIAPH2-AS1 knockdown.
Fig 4: Identification of miRNAs that likely regulates the expression of SLC7A11 and Pax3.(A) Microarray detection was performed to find the differentially expressed miRNAs between PE and normal placental tissues. (B) Bioinformatic analysis using TargetScan, miRmap, and miRanda predicted 607 miRNAs and 1421 miRNAs that target Pax 3 and SLC7A11, respectively. Venn diagram showed the intersection among the upregulated miRNAs and those targeting Pax 3 and SLC7A11. PCR was used to quantify the expression of five miRNAs shown in the intersection region of Venn diagram in normal and PE placental tissues (C) as well as miRNA-30b-5p expression in the normal and PE serum (D).
Fig 5: The regulatory effect of the HOXD8/DIAPH2-AS1/PAX3 network on cell proliferation and invasionEither HOXD8 or DIAPH2-AS1 were knocked down or PAX3 was overexpressed in HTR-8/SVneo cells under hypoxia. Afterward, HOXD8 and PAX3 expression was measured by Western blot assay (A). Cell viability was assessed using MTT testing agent (B). Cell migration and invasive capacities were evaluated by scratch (C) and transwell cell invasion (D) assays, respectively. *P<0.05, **P<0.01, and ***P<0.001. Nor: normoxia; LO: Low oxygen (hypoxia); HOX(-): HOXD8 knockdown; DIA(-):DIAPH2-AS1 knockdown; Pax3(+):Pax3 overexpression.
Supplier Page from Abcam for Anti-PAX3 antibody