Fig 2: Effects of Myrf on the activity of regulatory regions of OPC-specific and oligodendrocyte-specific target genes of Sox10. (A, B) Localization and distance of ECRs (open boxes) relative to the TSS (arrow) of the mouse Tgfb2, Id4, Wnt7a, Mag, Aatk and Mbp genes. Exons are depicted as black boxes. In case of Mag, the ECR corresponds to the promoter (prom). (C–J) Luciferase assays in N2a cells transiently transfected with reporter genes under control of regulatory regions from the Tgfb2 (C), Id4 (D), Wnt7a (E), Cspg4 (F), Pdgfra (G), Mag (H), Aatk (I) and Mbp (J) genes in the absence (–) or presence of Myrf, Sox10, Sox6 or a combination of Myrf and Sox protein. Reporter gene expression was determined in extracts 48 h after transfection and effector-dependent activation rates are presented as fold inductions ± SEM with transfections in the absence of effectors arbitrarily set to 1 for each reporter construct (n = 3–4). Differences were statistically significant as determined by one way Anova with Bonferroni correction (*P = 0.05; **P = 0.01; ***P = 0.001).

Fig 3: Structural requirements for Myrf function. (A) Schematic representation of the Myrf protein, its functional domains and the mutants used in the study. Numbers indicate positions of amino acids that define beginning or end of domains or constructs. P-rich, proline-rich domain; DBD, DNA binding domain; ICD, intracellular chaperone domain; CC, coiled coil domain; TM, transmembrane domain. (B, C) Luciferase assays in N2a cells transiently transfected with reporter genes under control of the Mag promoter (B) or Wnt7a regulatory region (C) in the absence (-) or presence of Myrf, various Myrf mutants, Sox10 and combinations thereof. Reporter gene expression was determined in extracts 48 h after transfection and effector-dependent activation rates are presented as fold inductions ± SEM with transfections in the absence of effectors arbitrarily set to 1 (n = 3). Differences were statistically significant as determined by one way Anova with Bonferroni correction (*P = 0.05; **P = 0.01; ***P = 0.001).

Fig 4: Wnt/ß-catenin signaling pathway is involved in hypoxia-induced C2C12 myoblast injury and is activated by hDPSC-CM. (A) Expression of ß-catenin, p-GSK-3ß (S9) and total GSK-3ß was detected using western blotting for 6, 12 and 24 h. Semiquantitative analysis of the expression ratios of (B) ß-catenin/ß-actin and (C) p-GSK-3ß/GSK-3ß. The levels of the downstream effectors of ß-catenin-related mRNAs, including (D) Lef1, (E) Tcf7, (F) Ccnd1 and (G) Ccna2, were assessed using quantitative PCR. (H) Expression of Wnt signaling ligands, including Wnt1, Wnt4 and Wnt7a. Semiquantitative analysis of the expression ratio of (I) Wnt1/ß-actin, (J) Wnt4/ß-actin and (K) Wnt7a/ß-actin. #P<0.05, ##P<0.01 and ###P<0.001 vs. N, *P<0.05 and ***P<0.001 vs. H. hDPSCs, human dental pulp stem cells; CM, conditioned media; p-GSK, phosphorylated-glycogen synthase kinase; Lef1, lymphoid enhancer binding factor 1; Tcf7, transcription factor 7; Ccnd1, cyclin D1; Ccna2, cyclin A2; N, normoxia; H, hypoxia.

Fig 5: Wnt7a can activated by the circRNA_001275/miR-370-3p axis. (A) Reverse transcription-quantitative PCR analysis of Wnt7a expression in circRNA_001275 and miR-370-3p transfection groups. (B) MTT assay results. **P<0.01 vs. pcDNA3.1 or si-NC. circRNA, circular RNA; DDP, cisplatin; miR, microRNA; NC, negative control; OE, overexpression vector; siRNA, small interfering RNA. Wnt7a, Wnt family member 7; OD, optical density.