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. 2022 Jan 18;132(2):e144579. doi: 10.1172/JCI144579

Figure 5. DKK1 rescued the loss of effect on OP recruitment and osteoclastic premetastatic niche formation following LGR4 knockdown.

Figure 5

(A and B) Knockdown of LGR4, transfection of DKK1 expression plasmid, or exogenous expression of DKK1 with LGR4 knockdown was performed in SCP46 cells, the conditioned medium was collected and injected i.p. into nude mice every day for 21 days of pretreatment, and tumor-free mice were euthanized at this time point. Representative images of IF double staining for RANK (green) and CD115 (red) and TRAP staining in the tibiae of mice (A), and quantitative analysis (B). White arrows indicate cells double-positive for RANK and CD115. Data indicate the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey’s post hoc test. n = 6 per group. Scale bars: 10 μm/5 μm (left/right, IF), 50 μm (TRAP). (C–F) SCP46-LUC cells were injected i.c. into tumor-free mice (tumor-bearing mice); 40 days later, the mice were sacrificed and bone analysis was performed. Representative images of bioluminescent, radiographic, H&E, TRAP, and IF double staining for RANK (green) and CD115 (red) in the tibiae of mice (C), and quantitative analysis (D–F). Yellow arrows indicate osteolytic lesions; red dotted lines indicate tumor zones; white arrows indicate cells double-positive for RANK and CD115. Data indicate the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, (D) 2-way ANOVA, (E) log-rank test, (F) 1-way ANOVA with Tukey’s post hoc test. n = 6 per group. Scale bars: 1 mm (micro-CT), 50 μm (H&E and TRAP), 10 μm/5 μm (left/right, IF).