Fig. 3. Effects of treatment of CoCl₂ on osteogenic differentiation.

(A) C3H/10T1/2 cells were pre-incubated with 0.1 mM CoCl₂ for 0 h, 24 h or 48 h. After incubation, the cells were replaced with osteogenic medium and cultured for 18 days, prior to staining with Alizarin red S. (B) Results from (A) were quantified by spectrophotometry. (C) C3H/10T1/2 cells were preincubated with 0.1 mM CoCl₂ for 0 h, 24 h, or 48 h. After incubation, cells were cultured with osteogenic medium for 3 days. Total cellular RNA was extracted, and gene expression of osteogenic markers was detected by semi-quantitative RT-PCR. Expression of actin was examined in the same sample as a control for the amount of present reverse-transcribed cDNA. (D) Effects of treatment of CoCl₂ during osteoblast differentiation. C3H/10T1/2 cells were pre-incubated with 0.1 mM CoCl₂ for 0 h, 24 h, or 48 h. After incubation, the cells were cells were replaced with an osteogenic medium and cultured for 10 days. At indicated times, total cellular RNA was extracted and gene expression of osteogenic markers was assessed by qRT-PCR. Values shown are normalized to β-actin levels. The data represent the mean±S.D. from triplicate independent experiments (^*p<0.05).