Expression of HES1 mRNA and protein in human osteosarcoma cell lines. HES1 mRNA in canine OSA cell lines and normal canine bone tissue (NB) expressed as 2(-ΔCT) normalized to HPRT1 (A). HES1 mRNA in human OSA cell lines and normal human osteoblasts expressed as 2(-ΔCT) normalized to the geometric mean of RBS15, GAPDH, 18SrRNA, and HPRT1 (C). Data are graphed as mean ± SEM, *** P < 0.001, ** P < 0.01, Two-way ANOVA with Dunnett’s Multiple Comparison Test. (B and D) Western blot shows characteristic distinct HES1 band at 30 kDa. Blot was stripped and re-probed with an antibody against α-tubulin to serve as a protein loading control.