Fig. 2: DDX1 interacts with EZH2 physically and is methylated at its lysine 234 site. | Nature Communications

Fig. 2: DDX1 interacts with EZH2 physically and is methylated at its lysine 234 site.

From: DDX1 methylation mediated MATR3 splicing regulates intervertebral disc degeneration by initiating chromatin reprogramming

Fig. 2: DDX1 interacts with EZH2 physically and is methylated at its lysine 234 site.

A Chordal graph and B GO biological process enrichment showing differential abundance of DDX1-interacting lysine methyltransferase in HEK-293T cells transfected with DDX1 WT. C CO-IP of the lysine methylation of exogenous DDX1 in HEK-293T (n = 3). D Interaction analysis between exogenous DDX1 and lysine methyltransferase (n = 3). E Protein level analysis of EZH2, β-actin was used as the loading control (n = 3, P = 0.0055). F IF of EZH2 and DDX1 in NP cells. Scale bar: 10μm (n = 3). G IHC of the EZH2 in NP. Scale bar: 100μm (n = 3). H Association assessment between the EZH2 and Pfirrmann grades (n = 3, P < 0.0001). I mRNA analysis of EZH2 in NP cells by RT-PCR (n = 3, P = 0.0009, P = 0.0021). J, K Co-IP of endogenous EZH2 with anti DDX1 antibody J and endogenous DDX1 with anti EZH2 antibody (K) in NP cells (n = 3). L, M Affinity pulldown using 6×His-tagged DDX1 followed by IB with anti-His and anti-GST antibodies, as well as CO-IP, were performed in HEK-293T cells. The latter was used to assess the binding between exogenously expressed EZH2 and either WT or mutant (KR, KM) forms of DDX1 under overexpression conditions (n = 3). N CO-IP was carried out in HEK-293T cells transiently transfected with the indicated constructs to characterize the interaction profiles between DDX1 and EZH2 (n = 3). O, P To investigate the regulatory effect of EZH2 on the lysine methylation of DDX1 under oxidative stress, CO-IP were conducted in TBHP-treated NP cells. Comparisons were made between EZH2-knockdown and EZH2-overexpressing conditions to determine the functional relevance of EZH2-mediated PTM (n = 3). Q CO-IP of the methylation of WT and K234R DDX1 in HEK-293T transfected with Flag-EZH2 (n = 3). Data information: *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± SEM. P value was quantified by Student’s t-test (B, E, I) and two-way ANOVA (H). Panels (C–G, J–Q) show the results of a representative similar result from one of three independent experiments.

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