Fig. 7: Suppression of osteoclastogenesis of HS-ICTO.

a Illustration of the transwell co-incubation experiment of K7M2 and RAW264.7 cells. The K7M2 cells were seeded in the upper chamber, and the RAW264.7 cells were seeded in the lower chamber. b The NFATc1, MMP-9, ACP5, and c-Fos gene expression of RAW264.7 cells co-incubated with K7M2 for 7 days measured by RT-qPCR (n = 3 biologically independent replicates). c Schematic depiction of the HS-ICTO inhibition mechanism affecting osteoclastogenesis. d Representative images of phalloidin-FITC stained RAW264.7 cells after different treatments. The large cells with green actin ring indicate osteoclasts. Positive control group (PC, only stimulated by 50 ng/mL RANKL). e Quantitative analysis of the size of osteoclasts (n = 30 cellular replicates). f Representative images of TRAP-stained RAW264.7 cells after different treatments. The fluorescence images in (d, f) were representative of three independently repeated experiments from each group. g Quantitative analysis of the TRAP-positive cell counts (n = 5 biologically independent replicates). h The NFATc1, MMP-9, ACP5, and c-Fos gene expression of RAW264.7 cells after different stimulations for 7 days measured by RT-qPCR (n = 3 biologically independent replicates). i The expressions of HIF-1α and α-tubulin of RAW264.7 were analyzed by western blot assay. Western blot experiments were independently repeated three times with similar results. Data are presented as mean ± SD, and ns represents no significant difference; in experiment (b), statistical significance was calculated using a two-tailed Student’s t-test and in experiment (e, g, h), statistical significance was calculated using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons; all tests were two-sided. Source data are provided as a Source Data file.