Fig. 6.

miR-378a and miR-140 synergistically regulated SSPC lineage fate via targeting the Pparα-Abca1 axis.

(A) Venn diagram of the miR-378a target gene. (B-C) Representative western blotting images of Bmp4, p38, Gata 4, Pparα, and Gapdh (B); quantification of Pparα expression (C) in SSPCs transfected with the mimic of miR-378a and its negative controls for 3 days. n = 3 per group. (D-E) Representative western blotting images of Pparα (D) and quantification of Pparα expression (E) in SSPCs transfected with the inhibitor of miR-378a and its negative controls for 3 days. n = 3 per group. (F) Schematic of miR-378a putative target sites in mouse Pparα 3′-UTR. CDS, coding sequence. (G) SSPCs were transfected with luciferase reporter carrying WT or MUT 3′-UTR of the Pparα gene (WT-Pparα-Fluc and/or Mut-Pparα-Fluc) and co-transfected with miR-NC and/or miR-378a, respectively. Fluorescence intensity was determined at 48 h after transfection; the asterisk means WT-Pparα-3′-UTR co-transfected with miR-NC vs. WT-Pparα-3′-UTR co-transfected with miR-378a (H-I) SSPCs were transfected with lenti-Pparα to induce osteogenesis and/or adipogenesis for 6 days. qRT-PCR analysis of the relative expression levels of osteogenic genes (H), n = 6 per group, and adipogenic genes (I), n = 3 per group. (J) Representative images of Alizarin red staining (J, top) and oil red O staining (J, bottom). Scale bar: 100 μm, n = 3 per group. (K) Schematic of protein interaction diagram of Pparα, Abca1, and Pgc-1α. (L) Representative western blotting images of Abca1 and Pgc-1 in SSPCs transfected with miR-140 and its negative controls. n = 3 per group. Data is shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant. (Non-parametric test is used in Alp expression of Fig. 6H, Welch's test is used in Sp7 expression of Fig. 6H, and student t-test is used in others).