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. Author manuscript; available in PMC: 2022 Mar 4.
Published in final edited form as: J Cell Physiol. 2020 Apr 23;235(11):8601–8612. doi: 10.1002/jcp.29704

FIGURE 4.

FIGURE 4

Cooperation of RUNX2 and EGR1 in Htra1 enhancers (a) Co-immunoprecipitation indicated a direct interaction between RUNX2 and EGR1. (b) Re-ChIP experiments were performed to determine whether RUNX2 and EGR1 were co-bound within Htra1-E4, E6, and E7. Relative luciferase activity of Htra1-E4 (c), E6 (d), and E7 (e) was detected after RUNX2 and EGR1 overexpression. RNA (f) and protein levels (g) of RUNX2, EGR1, HTRA1, OSX, OPN, and OCN in MC3T3-E1 cells with the individual or combined knockdown of RUNX2 and EGR1. Alizarin Red staining combined with alkaline phosphatase staining (h) showed calcified nodules and alkaline phosphatase activity (i) in individual or combined knockdown of RUNX2 and EGR1. All data are presented as means±SDs. *p < .05, **p < .01, ***p < .0001. ChIP, chromatin immunoprecipitation; EGR1, early growth response 1; Runx2, Runt-related transcription factor 2; SD, standard deviation