Fig. 3: Activation of the EZH2-dependent DDX1 methylation pathway promotes IVDD.

A, B The siControl and siEZH2 plasmids were transfected into TBHP-treated NP cells, and the Collagen II (P < 0.0001) and MMP3 (P = 0.0022) was measured (n = 3). C, D Cells derived from NP/siEZH2 cells overexpressed with indicated DDX1-WT or DDX1-K234M were used to detect Collagen II (P = 0.0194) and MMP3 (P = 0.0208) expression by western blot (n = 3). E, I Illustration presenting the experiment design. The LV-scrambled or LV-shEZH2 were used to knock down EZH2 in needle puncture model rats (scrambled group, n = 5 and shEZH2 group, n = 15). Subsequently, LV-DDX1 WT (DDX1 WT group, n = 5) and LV-DDX1 K234M (DDX1 K234R group, n = 5) were overexpressed in the shEZH2 group rats and injected weekly into the NP. The duration was 4 weeks. F–H X-ray F), CT (G), MRI results H) of rat vertebrae (n = 5). J, L-M) DHI (P < 0.0001, P = 0.0019), Pfirrmann grades (P = 0.0017, P = 0.0036) and histological points (P < 0.0001, P = 0.0001) of rat discs (n = 10). K HE and SO&FG of rat discs. Scale bar: 1 mm. N, O IHC of collagen II (P < 0.0001, P < 0.0001) and MMP3 (P < 0.0001, P = 0.0003) in rat discs. Scale bar: 150μm (n = 5). Data information: *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± SEM. P value was evaluated by Student’s t-test (B, D) and two-way ANOVA (J, L, M, O). Panels (A, C) show the results of a representative similar result from one of three independent experiments. Panels (F–H, K, N) show the results of a representative similar result from one of five independent experiments.