Figure 5.

Effects of NFATc1 knockdown in C3H10T1/2 cells under hypoxic conditions. NFATc1 expression was suppressed in pelleted C3H10T1/2 cells grown in pellets under hypoxic conditions using a double-stranded small interfering (si) RNA targeted to the murine NFATc1 mRNA sequence (si-NFATc1). A scrambled 19-mer siRNA with no homology to any known mouse sequence was used as the negative control (si-RNA-control). Expression of NFATc1 was determined at the mRNA level by RT-PCR (A) and protein level by western blot (B) at the time points indicated to determine the efficacy of the siRNA knockdown. The mRNA expression levels of (C) Sox9; (D) aggrecan; (E) collagen IIa1; and (F) collagen Xa1 were determined by RT-PCR in cells transfected with the si-NFATc1 or si-RNA-control sequences by RT-PCR at the times indicated. To confirm that NFATc1 bound to the promoter region of Sox9, a ChIP assay was performed pulling down the protein/nucleic acid complex using an anti-NFATc1 antibody or control rabbit IgG. (G) Representative image showing that DNA immune-precipitated with the anti-NFATc1 antibody contained a Sox9 fragment. The input lane was a 10-fold dilution of the cell lysate. (H) The difference in the amount of Sox9 that was pulled down with the NFATc1 or control IgG antibodies were quantified. The bars represent means ± SD (n=3). *0.05, compared with the IgG group.