Fig 2: NSUN5 negatively regulated CX3CL1 expression in m5C modification manner. (A) The transfection efficiency was measured by qPCR. (B) The levels of CX3CL1 mRNA were measured using qPCR. (C) The protein expression of NSUN5 in RANKL-stimulated RAW 264.7 cells were measured using Western blot. (D) The m5C modification level of CX3CL1 was detected by MeRIP. (E) The interaction between NSUN5 and CX3CL1 was measured by RIP. (F) The prediction of m5C modification sites in CX3CL1. (G) The binding relationship between NSUN5 and CX3CL1 was analyzed by Luciferase assay. (H) The stability of CX3CL1 mRNA was measured by qPCR after RAW 264.7 cells were treated with actinomycin D at 0, 6, 12, and 24 h. Data were analyzed by Student’s t-test (A, B, D, G), one-way ANOVA (E) or two-way ANOVA (H). All data are presented as the mean ± SD. *p < 0.05 and **p < 0.01. (n = 3).

Fig 3: CX3CL1 abrogated the bone-protective effects of NSUN5 in OVX-induced osteoporotic mice. (A-B) The effects of CX3CL1 and NSUN5 on (A) Micro-CT, (B) bone mineral density (BMD), (C) bone volume fraction (BV/TV), and (D) trabecular separation (Tb.Sp) of femurs. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test (B-D). All data are presented as the mean ± SD. **p < 0.01. (n = 6).