Fig. 4: Irgm1 mediates the changes in osteoclast marker genes by regulating ROS.

A WB analysis of Nfatc1, Traf6, and Ctsk in protein levels in osteoclasts derived from RAW264.7 cells after different stimulation for 3 days. B–D Quantitative analysis of Nfatc1 (B), Traf6 (C), and Ctsk (D) from (A) was normalized to GAPDH and presented as means ± SD (n = 3). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs the indicated groups. E–G RT-qPCR demonstrated the mRNA levels of Nfatc1 (E), Traf6 (F), and Ctsk (G) in osteoclasts with different treatments. Quantitative data were normalized to GAPDH and presented as means ± SD (n = 3), ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs the indicated groups. H DCFH-DA probe labeled intracellular ROS in RAW264.7. Cells from different groups treated with 40 ng/ml RANKL for 24 h were detected by DCFH-DA probe and visualized by fluorescence microscope. I, J Quantitative results from (H) (n = 3). ^∗∗∗p < 0.001 vs the indicated group. K Flow cytometry measured the density of DCFH-DA in RAW264.7 cells from different groups after 24 h of treatment. L, M Quantitative results from (K) (n = 3). ^∗p < 0.05 and ^∗∗∗p < 0.001 vs the indicated groups. N TRAP staining of osteoclast differentiated from RAW264.7. Cells from different groups were treated with or without 5 mM Nac for 5 days under 40 ng/ml RANKL conditions. Scale bar = 100 μm. (O) Quantitative analysis of TRAP-positive MNCs from (N) (n = 3). ^∗p < 0.05 and ^∗∗p < 0.01 vs the indicated group. P WB analysis of expression of Nfatc1, Traf6, and Ctsk in RAW264.7 from different groups of cells treated with or without 5 mM Nac for 3 days under 40 ng/ml RANKL conditions. Q–S Quantitative analysis of Nfatc1 (Q), Traf6 (R), and Ctsk (S) from (P) was normalized to GAPDH and presented as means ± SD (n = 3). ^∗p < 0.05 vs the indicated groups.