Figure 4
Silencing of Krox20 in preosteoclasts accelerates cell-cycle progression. (A) Preosteoclasts derived from WT, Krox20+/−, and Krox20−/− mice were plated in 12-well plates at 20 000 cells per well and cultured with 100 ng/mL M-CSF for 3 days before cell-cycle profiling. Bars represent percentage of cells in the S+G2/M phases. (B) Apoptosis was assessed in preosteoclasts obtained as in panel A using annexin V and propidium iodide staining. Survival corresponds to the percentage of cells negative for both FITC–annexin V labeling and propidium iodide. (A-B) Bars represent mean ± SEM; n = 4; #P < .05, 1-way analysis of variance for the effect of gene dosage. (C) Combined Krox20 immunohistochemistry (brown/black) and TRAP staining (purple) of a section from a WT distal femur, showing a representative Krox20-expressing osteoclast. Arrow points at a brown/black granular immunohistochemical reaction product indicating nuclear Krox20. Arrowheads depict faint brown cytoplasmic Krox20 immunostaining. (D) Splenocytes from WT mice were cultured for 3 days with 100 ng/mL M-CSF, and Krox20 expression was silenced using 2 different shRNA-coding lentiviruses (shKrox20-1 and shKrox20-2). Krox20 mRNA levels were determined by RT-qPCR and corrected for the corresponding expression of rpL10A. Bars represent expression levels relative to cells transduced with a nonspecific (NS) shRNA lentivirus. (E) Preosteoclasts were plated in 48-well format at 6000 cells per well and cultured with 100 ng/mL M-CSF, and Krox20 expression was silenced as in panel D. Proliferation was determined between days 4 and 9 after transduction using MTT assay. (F) Preosteoclasts were plated in 12-well plates at 20 000 cells per well and cultured with 100 ng/mL M-CSF. Cells were then transduced with lentiviruses encoding the shKrox20-2 or the NS shRNA, and cell-cycle analysis was performed as in panel A. Representative cell-cycle profiles are shown on the right. (G) Apoptosis was assessed in preosteoclasts obtained as in panel F. (D-G) Bars represent mean ± SEM; n = 4; *P < .05 vs NS.

Silencing of Krox20 in preosteoclasts accelerates cell-cycle progression. (A) Preosteoclasts derived from WT, Krox20+/−, and Krox20−/− mice were plated in 12-well plates at 20 000 cells per well and cultured with 100 ng/mL M-CSF for 3 days before cell-cycle profiling. Bars represent percentage of cells in the S+G2/M phases. (B) Apoptosis was assessed in preosteoclasts obtained as in panel A using annexin V and propidium iodide staining. Survival corresponds to the percentage of cells negative for both FITC–annexin V labeling and propidium iodide. (A-B) Bars represent mean ± SEM; n = 4; #P < .05, 1-way analysis of variance for the effect of gene dosage. (C) Combined Krox20 immunohistochemistry (brown/black) and TRAP staining (purple) of a section from a WT distal femur, showing a representative Krox20-expressing osteoclast. Arrow points at a brown/black granular immunohistochemical reaction product indicating nuclear Krox20. Arrowheads depict faint brown cytoplasmic Krox20 immunostaining. (D) Splenocytes from WT mice were cultured for 3 days with 100 ng/mL M-CSF, and Krox20 expression was silenced using 2 different shRNA-coding lentiviruses (shKrox20-1 and shKrox20-2). Krox20 mRNA levels were determined by RT-qPCR and corrected for the corresponding expression of rpL10A. Bars represent expression levels relative to cells transduced with a nonspecific (NS) shRNA lentivirus. (E) Preosteoclasts were plated in 48-well format at 6000 cells per well and cultured with 100 ng/mL M-CSF, and Krox20 expression was silenced as in panel D. Proliferation was determined between days 4 and 9 after transduction using MTT assay. (F) Preosteoclasts were plated in 12-well plates at 20 000 cells per well and cultured with 100 ng/mL M-CSF. Cells were then transduced with lentiviruses encoding the shKrox20-2 or the NS shRNA, and cell-cycle analysis was performed as in panel A. Representative cell-cycle profiles are shown on the right. (G) Apoptosis was assessed in preosteoclasts obtained as in panel F. (D-G) Bars represent mean ± SEM; n = 4; *P < .05 vs NS.

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