Prdm5 Promotes the Transcription of ECM Genes Figure 2. Prdm5 deregulation impairs osteogenic differentiation in vitro. A) Upper panel. qRT-PCR of Prdm5 levels in MC3T3 cells transduced with lentiviral shRNA constructs against Prdm5 (shPrdm5-a and shPrdm5-b) and control construct. Lower panel. Prdm5 western blot from the same experiment. Tubulin is included as loading control. B) Upper panel. Quantification of Alizarin red staining after 21 days of osteogenic differentiation in MC3T3 cells. Data are presented as mean of 3 independent experiments 6 SEM. ** = p,0.01 and *** = p,0.005 (t-test). Lower panel. Representative image from osteogenic differentiation experiments. C) Western blot showing overexpression of human PRDM5 (marked with star) in MC3T3 cells (filled circle = endogenous Prdm5). GFP overexpression is used as a negative control and Vinculin western blot for equal protein loading. D) Quantification of Alizarin red staining from osteogenic differentiation experiments of MC3T3 cells overexpressing GFP or PRDM5. A representative experiment is shown and data are presented as average 6 standard deviation. E) qRT-PCR analysis of WT and Prdm5LacZ/LacZ calvaria osteoblasts for PLoS Genetics | www.plosgenetics.org 4 May 2012 | Volume 8 | Issue 5 | e1002711