Fig. 7

Inhibitors of Prmt1 attenuated CHOM progression. (A) Experimental design schematic showing the four groups of mice: Control, CHOM, TCE-5003-treated, and MS023-treated. (B–D) Serum cytokine levels of IL-1β (B), IL-6 (C), and TNF-α (D) in Control, CHOM, TCE-5003, and MS023 groups (n = 8 per group). (E) Representative images of tibial inflammatory lesions in Control, CHOM, TCE-5003, and MS023 groups. (F) Representative H&E-stained sections of tibiae from Control, CHOM, TCE-5003, and MS023 groups. Asterisks (*) indicated areas of sequestrum formation. (G) Quantification of bacterial load in tibiae. Homogenates from 1 g of tibial tissue were plated on TSA plates, incubated at 37 °C for 20 h, and CFUs were enumerated. (H–K) mRNA expression levels of Adamts3(H), Adamts8(I), Adamts12(J), and Adamts14(K) in tibial tissues from Control, CHOM, TCE-5003, and MS023 groups (n = 8 per group). (L and M) Western blot analysis of Ncoa4, Adamts3, and Adamts8 (L), as well as Adamts12 and Adamts14 (M) protein levels in tibial tissues from Control, CHOM, TCE-5003, and MS023 groups (n = 3 per group). (N) Total protein methylation levels in tibial tissue homogenates from Control, CHOM, TCE-5003, and MS023 mice. Homogenates were prepared by mixing three independent tibiae of equal weight from the same groups of mice. Left panel: CBB staining confirmed equal total protein loading. Right panel: Immunoblot analysis revealed global protein methylation levels. (O) Effects of TCE-5003 and MS023 treatment on Ncoa4 methylation and ubiquitination levels. Tibial tissues from control, CHOM, TCE-5003, and MS023 groups (n = 3 per group; tissues from each group were pooled in equal weights prior to the IP assay) were subjected to immunoprecipitation using anti-Ncoa4-conjugated agarose beads. The immunoprecipitated proteins were analyzed by immunoblotting with anti-Ncoa4, anti-ADMA, and anti-Ubiquitin antibodies. *P < 0.05; **P < 0.01; ***P < 0.001