Fig. 3

miR-1323 directly suppresses BMP4 and SMAD4 levels. a, b Human mesenchymal stromal cells were placed in osteogenic differentiation medium and transfected with miR-1323 mimic or inhibitor. After 7 days of culture, BMP4 and SMAD4 levels were measured by Western blot. c (Top) Wild-type miR-1323 binding site in the BMP4 3′UTR luciferase reporter construct (WT-BMP4 3′UTR) and (bottom) the engineered mutant miR-1323 binding site in the BMP4 3′UTR luciferase reporter construct (MUT-BMP4 3′UTR). d Luciferase reporter activity levels in HEK293 cells transfected with miR-1323 mimics or inhibitor along with the WT-BMP4 3′UTR or MUT-BMP4 3′UTR luciferase reporter construct. e, f Luciferase reporter activity levels in HEK293 cells transfected with miR-1323 mimics or inhibitor along with (e) the wild-type SMAD4 3′UTR luciferase reporter construct (WT-SMAD4 3′UTR) or mutant SMAD4 luciferase reporter construct (MUT-SMAD4 3′UTR) for site 1 or (F) WT-SMAD4 3′UTR or MUT-SMAD4 3′UTR for site 2. See Additional figure 1 for wild-type and engineered mutant site 1 and site 2 in WT-SMAD4 3′UTR and MUT-SMAD4 3′UTR. *p < 0.05, **p < 0.01 [miR-1323 inhib vs. NC inhib; miR-1323 mimics vs. NC mimics]. Data presented as means ± SEMs. All in vitro experiments: 3 biological replicates × 3 technical replicates