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. 2024 Aug 18;27(9):110734. doi: 10.1016/j.isci.2024.110734

Figure 7.

Figure 7

Validation of the effects of key translational factors on regulating osteoclastogenesis of in vitro BMMs

See also Figure S4 and Table S12. The relative expression of TRAP and CTSK in scramble or knock-down OCs, related to Figure 7A left, Table S13. The relative expression of TRAP and CTSK in scramble or knock-down OCs, related to Figure 7A right, Table S14. Area of single resorption pit on bone slices (scramble and knock-down OCs), related to Figure 7B, Table S15. TRAP positive cells in 4 TFs knock-down in vitro cultured BMMs of 20 m and 6 m mice, related to Figure 7D.

(A) The expression of TRAP and CTSK, the canonical makers of mature OC, were measured by RT-qPCR before and after 3 days and 8 days the stimulation of RANKL, which are sequential time points along the differentiation of OC. The expression of TRAP and CTSK increased in TFs knockdown BMMs of 6 m and decreased in TFs knockdown BMMs of 20 m mice after 8 days stimulation of RANKL. The two upper plots showed the expression of CTSK and the lower plots showed the expression of TRAP; the two plots on the left demonstrated results from 20 m mice while the other two plots on the right showed the results from 6 m mice.

(B) Pits array analysis by SEM scanning showed smaller pits formed by aged (20 m) TF knockdown BMMs and bigger pits formed by young (6 m) TF knockdown BMMs.

(C–E) Images and representative inserts of TRAP staining of in vitro-cultured OC. TFs knockdown BMMs generated more TRAP positive cells in young (6 m) BMMs after Irf8, Sox4, and Klf4 were knocked down. On the contrary, aged (20 m) BMMs couldn’t form mature OCs after Cebpd was blocked. Data are shown as mean ± s.e.m. In (A), n = 116 (26 Scr, 36 Cebpd, 18 Sox4, 18 Irf8, 18 Klf4) for TRAP group; n = 116 (26 Scr, 36 Cebpd, 18 Sox4, 18 Irf8, 18 Klf4) for CTSK group, each dot represents a single individual. In (B), n = 10 (1 Scr, 2 Cebpd) for 20 m group; n = 7 (1 Scr, 2 Sox4, 2 Irf8, and 2 Klf4) for 6 m group. In (D), n = 9 (3 Scr, 6Cebpd) for 20 m. In (E), n = 28 (4 Scr, 8Sox4, 8 Irf8, 8 Klf4) for 6 m, each dot represents a single individual. p values were determined by two-tailed Student’s t test. “Scr” stands for scramble.