Figure 3
Figure 3. Increased proliferation in osteoclast precursors derived from Krox20+/− mice. (A) Splenocytes from WT and Krox20+/− newborn mice were cultured for 3 days with 100 ng/mL M-CSF. Krox20 and Krox24 mRNA expression in subconfluent preosteoclasts cultures was assessed by RT-qPCR and corrected for the expression of rpL10A. (B) Spleen-derived preosteoclasts as in panel A were plated in 96-well plates at 2000 cells per well and cultured with 100 ng/mL M-CSF. Medium was changed on days 3 and 5, and proliferation was determined after 7 days using MTT assay. (C) Splenocytes from WT and Krox20+/− newborn mice were plated in 96-well plates at 150 000 cells per well and cultured from the first day with 20 ng/mL M-CSF and 100 ng/mL RANKL. Medium was changed on days 3 and 5, and TRAP staining was performed on day 8. Bars represent relative TRAP+ area. (D-E) Spleen-derived preosteoclasts pretreated with 100 ng/mL M-CSF alone as in panel A were plated in 96-well plates at 5000 cells per well and cultured with 20 ng/mL M-CSF and 100 ng/mL RANKL for 7 days. Medium was changed on days 3 and 5. Bars represent relative TRAP+ area. (E) Representative images of TRAP-positive osteoclasts derived from WT (left) and Krox20+/− (right) mice; bar = 0.5 mm. (F) Krox20 and Krox24 mRNA in primary calvarial osteoblastic cultures from Krox20+/− and control mice was assessed by RT-qPCR and corrected for the expression of rpL10A. (G-H) Primary calvarial osteoblast cultures from WT and Krox20+/− mice were fixed and stained with alizarin red 20 days after confluence. Bars represent percent alizarin red-positive area. (H) Representative images from WT- (left) and Krox20+/−-derived (right) cultures. (I-J) Bone marrow–derived cultures from WT and Krox20+/− mice. Number of alizarin red-positive CFU-Ob colonies (I) and mean colony size (J) were determined in day-20 cultures after fixation and staining. Bars represent mean ± SEM of Krox20+/+ (white) and Krox20+/− (gray) cells. *P < .05 compared with control littermates. Assays were performed in triplicate per animal, and the number of animals is specified in parentheses inside each bar.

Increased proliferation in osteoclast precursors derived from Krox20+/− mice. (A) Splenocytes from WT and Krox20+/− newborn mice were cultured for 3 days with 100 ng/mL M-CSF. Krox20 and Krox24 mRNA expression in subconfluent preosteoclasts cultures was assessed by RT-qPCR and corrected for the expression of rpL10A. (B) Spleen-derived preosteoclasts as in panel A were plated in 96-well plates at 2000 cells per well and cultured with 100 ng/mL M-CSF. Medium was changed on days 3 and 5, and proliferation was determined after 7 days using MTT assay. (C) Splenocytes from WT and Krox20+/− newborn mice were plated in 96-well plates at 150 000 cells per well and cultured from the first day with 20 ng/mL M-CSF and 100 ng/mL RANKL. Medium was changed on days 3 and 5, and TRAP staining was performed on day 8. Bars represent relative TRAP+ area. (D-E) Spleen-derived preosteoclasts pretreated with 100 ng/mL M-CSF alone as in panel A were plated in 96-well plates at 5000 cells per well and cultured with 20 ng/mL M-CSF and 100 ng/mL RANKL for 7 days. Medium was changed on days 3 and 5. Bars represent relative TRAP+ area. (E) Representative images of TRAP-positive osteoclasts derived from WT (left) and Krox20+/− (right) mice; bar = 0.5 mm. (F) Krox20 and Krox24 mRNA in primary calvarial osteoblastic cultures from Krox20+/− and control mice was assessed by RT-qPCR and corrected for the expression of rpL10A. (G-H) Primary calvarial osteoblast cultures from WT and Krox20+/− mice were fixed and stained with alizarin red 20 days after confluence. Bars represent percent alizarin red-positive area. (H) Representative images from WT- (left) and Krox20+/−-derived (right) cultures. (I-J) Bone marrow–derived cultures from WT and Krox20+/− mice. Number of alizarin red-positive CFU-Ob colonies (I) and mean colony size (J) were determined in day-20 cultures after fixation and staining. Bars represent mean ± SEM of Krox20+/+ (white) and Krox20+/− (gray) cells. *P < .05 compared with control littermates. Assays were performed in triplicate per animal, and the number of animals is specified in parentheses inside each bar.

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