Figure 5.

BMAL1 regulates sequential chondrocyte differentiation through directly activating Ptch1 and Ihh transcription. A, B, The transcription factor BMAL1 bound to the Ptch1 and Ihh in primary mandibular condylar chondrocytes. Chromatin immunoprecipitation assays were performed using anti‐BMAL1 with anti‐IgG as a negative control (n = 3 for each bar). Data represented as mean ± SEM. ^*** P < .001. C, D, Dual‐luciferase assays were performed to measure the activities of Ptch1 and Ihh promoters in primary mandibular condylar chondrocytes. Filled blue circle, BMAL1‐binding site. Filled crossed blue circle, mutant of BMAL1‐binding site (n = 3 for each bar). Data represented as mean ± SEM. ^* P < .05, ^** P < .01, ^*** P < .001. E, PTCH1 overexpression was assessed by Western blot. F‐N, The cell proliferation and cell apoptosis were detected by CCK‐8 (F and I), EdU staining (G, H, I, J) and flow cytometry assays (L‐N) in BMAL1 knockdown with PTCH1 overexpressing or SAG supplemented primary mandibular condylar chondrocytes (n = 3 for each bar). Data represented as mean ± SD. ^*** P < .001