Figure 1.

Bioinformatics prediction and basal expression of the candidate transcription factors in MCT cells. (A) Two putative and overlapping DLX5 bindings sites within the 150-bp Col10a1 cis-enhancer were predicted by JASPAR. These two sites are at the 3-prime of the enhancers with the relative profile score threshold over 80%. (B) The predicted DLX5 binding site by JASPAR within the Col10a1 enhancer showed four bases overlapping with the previously identified tandem-repeat RUNX2 sites (TGTGGGTGTGGC).²⁶(C) MCT cells were cultured at 32 °C for 3 days (proliferative chondrocytes) and induced to hypertrophy at 37 °C for 24 h (hypertrophic chondrocytes). The mRNA expression of Col10a1, Dlx5, Hoxa3, and Nrf2 was detected by qRT-PCR. Col10a1 mRNA expression was significantly increased in hypertrophic MCT cells as expected. The up-regulation of mRNA levels of Dlx5, as well as two other candidate transcription factors Hoxa3 and Nrf2, were tested and confirmed in hypertrophic MCT cells. (D) MCT cells were cultured at 32 °C for 3 days (proliferative chondrocytes) and induced at 37 °C for 48 h (hypertrophic chondrocytes). The protein expression of COL10A1 and DLX5 was detected by Western blot. Data were expressed as mean ± SEM from three independent experiments. ∗P < 0.05, ∗∗P < 0.01.