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. 2019 Jul 25;28(15):1004–1014. doi: 10.1089/scd.2019.0124

FIG. 3.

FIG. 3.

SFRP1 knockdown enhances osteo-differentiation in PDL fibroblasts by increasing H3K4me3 marks on RUNX2 and SP7 promoters. PDL cells stably expressing a con sh or shRNA against SFRP1 (SFRP1 sh1 and SFRP1 sh2) were grown with or without osteogenic induction. ALP activity was assayed after 7 days (A) and mineral deposits stained using ARS after 14 days (B). Note the elevated ALP activity in SFRP1 sh2 expressing PDL cells grown under nonmineralizing conditions. (C) mRNA expression of mineralization markers in SFRP1 sh1 expressing cells compared against con sh expressing cells. (D) Western blot analysis for osteogenic markers in lysates from con sh and SFRP1 sh1 expressing cells. (E) β-catenin enrichment on its response element located on the RUNX2 promoter in con sh and SFRP1 sh1 expressing cells. Relative enrichment of H3K4me3 and H3K27me3 histone modifications on the RUNX2 promoter (F) and on the SP7 promoter (G) in con sh and SFRP1 sh1 expressing cells. ChIP regions analyzed are marked as base pairs upstream to the TSS for respective promoters. Semiquantitative real-time RT-PCR for mRNA expression and ChIP analysis data are representative of three independent experiments with similar results. (Statistical significance is depicted as *P < 0.05, **P < 0.01, ***P < 0.001). con sh, control shRNA; RT-PCR, real-time polymerase chain reaction; shRNA, short hairpin RNA.