Fig. 1

Knockdown of PUM2 promotes the in vitro osteogenic potential of MSCs. A Representative images of western blot analysis for protein levels of PUM1, PUM2, RUNX2, COL1A1, and OPN in MSCs that were cultured with growth medium (GM) or osteogenic differentiation medium (ODM) for 10 days. β-ACTIN was used as a loading control. B qRT-PCR analysis of PUM1 and PUM2 mRNA expression in MSCs that were transfected with non-targeting control (NC), PUM1, or PUM2 siRNA. *P < 0.05 in comparison with NC group (n = 3 experimental replicates). C Representative images of alizarin red S staining in NC, PUM1, or PUM2 siRNA-transfected MSCs cultured with ODM for 10 days. For quantification, absorbance was measured at 595 nm following destaining with 10% cetylpyridinium for 30 min. *P < 0.05 compared with NC group (n = 3 experimental replicates). D Fluorescence and western blot images showing the activity of OCN promoter and the OCN promoter reporter quantity. MSCs were transduced with lentivirus of OCN pGreenZeo differentiation reporter, the transduced MSCs were then transfected with NC, PUM1, or PUM2 siRNA. The bar graph indicates the fluorescence-based activities of the OCN promoter. *P < 0.05 compared with the NC group (n = 3 experimental replicates). OCN, osteocalcin; copGFP, copepod green fluorescence protein. Scale bar = 150 μm. E ALP staining was performed to evaluate whether the PUM2 siRNA-transfected MSCs are committed to the osteogenic lineage. Staining was performed using undifferentiated MSCs on day 4 post-transfection. This experiment was performed (n = 3 experimental replicates), and representative data are shown. F Likewise, the MSCs, on 4 days of post-transfection, were harvested to measure ALP activity. The results for ALP activity were normalized to the amount of total protein. *P > 0.05 compared with NC (n = 3 experimental replicates)