Fig. 4

PUM2 downregulated the activity of DLX5 3’-UTR reporter in MSCs. A Schematic diagram of the experimental method showing the activity of human DLX5 3’-UTR downstream of a CMV-driven reporter (GFP) by PUM2. B Protein levels of PUM2 and GFP in MSCs transduced with DLX5 3’-UTR reporter-GFP vector. Cells were selected using puromycin (10 µg/ml) and then transfected with NC or PUM2 siRNA before western blot to compare the GFP activities. *P < 0.05 or *P < 0.001 compared with the NC (n = 3 experimental replicates). C, D Protein levels of PUM2::HA, PUM2::GFP, and DLX5 3’UTR::GFP in MSCs transduced with the DLX5 3’-UTR reporter-GFP vector. The cells were selected using puromycin (10 µg/ml) and transfected with the pcDNA or pEGFP-C1 vector control, pcDNA-PUM2-HA, pEGFP-C1-PUM2, or pEGFP-C1-ΔPUM2-HD vector; cells were analyzed by western blot to compare the GFP activities. **P < 0.01 or ***P < 0.001 compared with the pcDNA or pEGFP-C1 vector control (n = 3 experimental replicates). E Schematic diagram of point mutation of the GFP/DLX5 mRNA 3’-UTR construct. To determine if PUM2 actually binds to the putative sequences [UGUA(N)AUA] within the 3’-UTR region of DLX5 mRNA, the DLX5 3’-UTR GFP reporter assay was performed using mutated constructs. F Protein levels of PUM2::HA and DLX5 3’UTR::GFP in MSCs transduced with the wild-type DLX5 3’-UTR reporter-GFP vector or the mutant DLX5 3’-UTR reporter-GFP vector. The cells were selected using puromycin (10 µg/ml) and transfected with the pcDNA vector control or pcDNA-PUM2-HA vector; cells were analyzed by western blot to compare the GFP activities. **P < 0.01 or ***P < 0.001 compared with the pcDNA vector control (n = 3 experimental replicates)