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. 2022 Jun 27;13:3684. doi: 10.1038/s41467-022-31356-7

Fig. 6. AP2α bridges the interaction between CIITA and the promoters of TNFSF11 and SOST in osteocytes.

Fig. 6

a Immunoprecipitates pulled down by c-myc-CIITA in osteocytes transfected with a c-myc–CIITA plasmid using Coomassie blue staining. b Pull-down of AP2α with c-myc-CIITA in HEK293T cells. c Co-immunoprecipitation of CIITA with AP2α in osteocytes transfected with c-myc-CIITA plasmid. d Co-immunoprecipitation of CIITA with AP2α in MLO-Y4 and MLO-A5 osteocytes cultured without or with 1 mM 2DDR. e, f Schematic of the Tnfsf11 (e) and Sost (f) promoter luciferase reporter. Solid boxes: promoter region; red crosses: mutation sites. The luciferase activity of Luc-Tnfsf11 or Luc-Sost constructs was set at 1. ns, not significant. g–h Western blot shows the expression of AP2α (g) and H3K14ac (h) in MLO-Y4 or MLO-A5 cells transfected with non-targeted shRNA (shCtrl) or Ap2α shRNA (shAp2α) cultured without or with 1 mM 2DDR. (GAPDH and H3: protein loading controls). i ChIP assay shows the enrichment of H3K14ac and CIITA in the promoter of Tnfsf11 or Sost genes in MLO-Y4 and MLO-A5 cells expressing shCtrl or shAp2α cultured without or with 1 mM 2DDR. Data in (e), (f), and (i) are presented as mean ± SD, n = 3 biological replicates. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test. Data shown in (a–d), (g), and (h) are representative of two independent experiments.