Fig. 1: CK2 is required for BMSC osteoblast commitment.

a Immunohistochemistry (IHC) for RUNX2 in human HO tissue. IgG was used for negative staining. Scale bar, 100 μm. b–d Human BMSCs expressing control shRNA (shCtrl) or shRNAs targeting CSNK2A1, -2A2, or -2B (shCSNK2A1, shCSNK2A2, shCSNK2B) were cultured under osteogenic conditions. ALP activity was examined at day 7 (b), mineralization activity was assessed by alizarin red staining at day 14 (c), and expression of osteogenic genes was assessed by RT-PCR at day 12 (d) after osteogenic culture. b n = 14; c, n = 8; d, n = 4 biologically independent samples. shCtrl or shCSNK2B-expressing human BMSCs (e) and shCtrl or shCsnk2b-expressing C3H10T1/2 cells (f) were transfected with the RUNX2-responsive reporter gene (OG2-luc) and Renilla in the absence (e) or in the presence (f) of RUNX2 overexpression. Three days after osteogenic culture (e) or 2 days after transfection (f), OG2-luc activity was measured and normalized to a Renilla. (n = 3 biologically independent samples). g Diagram depicting kinetics of CK2 expression over the differentiation of human BMSCs. h, i shCtrl or shCSNK2B-epxressing human BMSCs were cultured under chondrogenic conditions for 21 days. After alcian blue staining, the size of chondrocyte pellets was measured (h). mRNA levels of chondrogenic genes were assessed by RT-PCR (i). Scale bar, 300 μm (h). h n = 5 (shCtrl) or 6 (shCSNK2B); i n = 4 biologically independent samples. j, k shCtrl or shCSNK2B-epxressing human BMSCs were cultured under adipogenic conditions for 12 days. Cells were stained with oil red O (j) and mRNA levels of adipogenic genes were assessed by RT-PCR (k). Scale bar, 100 μm (j). k n = 4 biologically independent samples. Data are representative of two (a) or three (b–f, h–k) independent experiments. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (a) and a two-tailed unpaired Student’s t test for comparing two groups (c–f, h, i, k; error bars, SD of biological replicates).