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. 2023 Mar 23;26(4):106470. doi: 10.1016/j.isci.2023.106470

Figure 3.

Figure 3

Osteoclast formation in ex vivo cultures elicits osteoprogenitor proliferation

(A) Representative phase contrast images of TRAP stained marrow explants following 4 days with (Dox.), without (Unind.) or with induction in the presence of OPG (Dox.+OPG). Arrow denotes lesion of cells, Arrowheads denote TRAP+, multinucleated osteoclasts, which appear dark in phase contrast.

(B) ELISA evaluation of soluble LIF in the culture media of marrow explants without (Unind.), with 4 days induction (Dox.) or 4 days induction and 100 ng/mL OPG (OPG). (n = 6 for OPG or n = 7 for Unind. and Dox.).

(C) Representative immunofluorescence images of the preosteoblast marker Runx2, the proliferation marker Ki67 and the nuclear stain Hoechst in cultures treated as in (A).

(D) Quantification of the fraction of Runx2+ preosteoblasts that exhibit staining for the proliferation marker Ki67. (n = 3).

(E) qPCR evaluation of the osteogenic markers Sp7, Dlx5 and Bglap under uninduced (black), Dox. induced (gray) or Dox. induced +100 ng/mL OPG (white) conditions. Fold expression is relative to 18s rRNA. (n = 5).

(F) Representative immunofluorescence images of the preosteoblast marker Runx2, the proliferation marker Ki67 and the nuclear stain Hoechst in MC3T3-E1 clone 14 preosteoblasts treated with (OPG) or without (control) 100 ng/mL OPG.

(G) Quantification of the fraction of Runx2+ preosteoblasts that exhibit staining for the proliferation marker Ki67 treated as in (F). (n = 3 for clone 4(circle), n = 3 for clone 14(square). Bar denotes summary average of both clones 4 and 14).

(H) qPCR evaluation of the osteogenic markers Sp7, Dlx5 and Bglap in clones 4 and 14 under control (black), 100 ng/mL OPG (white) conditions. Fold expression is relative to 18s rRNA. (n = 3) Error bars = ±SEM. Significance was assessed via paired t-test, where ∗p=<0.05, ∗∗p=<0.01, ∗∗∗p=<0.001.