Fig 1: The cluster miR-424(322)/503 reduces adipogenesis. A) Human miR-424-5p and miR-503-5p share common target genes with mouse miR-322-5p and miR-503-5p. B) Heatmap illustrating representative enriched pathways from pre-selected human and mouse miRNA cluster targets based on AT RNA sequencing. C) Left, bright field, and oil-red representative images of in vitro differentiation of human adipocytes. Right, expression of miR-424(322) and miR-503 in differentiating human preadipocytes (PA) at day 7 (Ad) and 14 (MA) after inducing adipogenesis, as explained in the Experimental Section of this article. D) Bisulfite sequencing analysis of 18 CpG in human preadipocytes and mature adipocytes. Total CpGs analyzed >2000. Upper figure illustrates the genomic location of the miR-424(322)/503 cluster. Green bar under miR-424 and miR-503 represents the genomic region analyzed that contains the 18 sequential CpG islands, three of them located within the pre-miR-424 (CpG 1 to 3, circled in blue). Middle graph contains all biological replicates (rows). Each row/replicate encompasses the 18 CpG dinucleotides and represents the average of =10 sequenced alleles (see in Figure S3C, Supporting Information). Percentage of methylation for each CpG is color-coded. Left scatter plot graph shows the percentage of methylation of each replicate (average of 18 CpG) across PA (n = 5) and MA (n = 6). E) Human preadipocytes and mature adipocytes challenged with 10 ng mL-1 of recombinant transforming growth factor beta (TGFß) for 14 days displayed enhanced expressions of miR-424(322) and miR-503, and impaired differentiation. Arrowheads indicate lipid droplets stained with Oil Red O (n = 6 biological replicates/group). F) Mouse embryonic fibroblasts (MEF) from miR-KO mice had increased adipocyte formation (Oil Red O staining) when cultured in an adipogenic medium. G) Analysis of markers of adipogenesis (i.e., Pparg, Cebpa, Fabp4, and adiponectin) by western blot in Wt and miR-KO adipocytes. H) Expression of a battery of genes related to the adipogenic program in mouse adipocytes derived from miR-KO (red) and Wt (green) MEF, the latter taken as reference (100%) (see also Figure S4B, Supporting Information). I) Different stages of the differentiation of human preadipocytes were challenged by our transient doxycycline (Dox)-inducible pTRIPz lentiviral system (pT-miR-424/503). PM-1, DM-2, AM-1, and BM-1 stand for preadipocyte, adipocyte differentiation, adipocyte maintenance, and adipocyte basal media, respectively. J,K) 14 day-lasting Dox-treated pT-miR424/503 human adipocytes (n = 6) exhibit impaired adipogenesis when compared to control cells (pT-Control, n = 4), as revealed by Oil Red O staining and protein levels of markers of adipogenesis (n = 2/group), and L) gene expression levels (n = 4/group), respectively (see also Figure S4C–G, Supporting Information). Data are presented as mean ± S.E.M. Statistical significance was determined by paired Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant.