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. 2020 Sep 16;22:1078–1091. doi: 10.1016/j.omtn.2020.09.014

Figure 3.

Figure 3

KDM2A Binds to Transcription Factor E2F1 and Inhibits Its Transcriptional Activity, Thereby Impeding Chondrocyte Proliferation In Vitro

(A) Venn diagram of the predicted downstream transcription factors of KDM2A by the hTFtarget and RNAInter websites with the genes related to knee OA in the GeneCards database. (B) Targeting relationship between KDM2A and the transcription factor E2F1 verified by the hTFtarget website. (C) Immunohistochemistry of E2F1 protein in articular cartilage tissues of OA (N = 54) and non-OA (N = 36) subjects. ∗p < 0.05 compared with articular cartilage tissues of non-OA subjects. (D) KDM2A mRNA expression determined by qRT-PCR in chondrocytes overexpressing KDM2A. ∗p < 0.05 compared with overexpression of NC (oe-NC)-transfected chondrocytes. (E) Representative western blots of E2F1 protein and its quantitation in chondrocytes overexpressing KDM2A. ∗p < 0.05 compared with oe-NC-transfected chondrocytes. (F) Endogenous E2F1 in Myc-labeled, KDM2A-treated chondrocytes. Input is a positive control, and IgG is a negative control. (G) Representative western blots of total E2F1 protein and its quantitation in chondrocytes treated with CHX (100 ng/mL) at 0 h, 6 h, 12 h, and 24 h. ∗p < 0.05 compared with oe-KDM2A-treated chondrocytes. ∗p < 0.05 compared with control chondrocytes. (H) GST-KDM2A fusion protein and E2F1 protein translated in vitro were used to perform a GST pulldown assay to detect exogenous E2F1. Input is a positive control, and GST is a negative control. (I) In the luciferase assay, cells of each group were treated with different doses of KDM2A to inhibit E2F1-mediated E2.Luc transcription in a dose-dependent manner. ∗p < 0.05. (J) KDM2A mRNA expression determined by qRT-PCR in chondrocytes overexpressing KDM2A or combined with E2F1. (K) Representative western blots of E2F1 protein and its quantitation in chondrocytes overexpressing KDM2A or combined with E2F1. ∗p < 0.05 compared with oe-NC-transfected chondrocytes; #p < 0.05 compared with oe-KDM2A-transfected chondrocytes; &p < 0.05 compared with oe-E2F1-transfected chondrocytes. (L) Proliferation of chondrocytes overexpressing KDM2A or combined with E2F1 measured by the CCK-8 assay. (M) The number of migrated chondrocytes overexpressing KDM2A or combined with E2F1 measured by the Transwell assay. (N) Representative images of migrating chondrocytes (×200). Data are shown as mean ± standard deviation of three technical replicates. Unpaired t test was applied for comparison between two groups. Data comparison among multiple groups was performed using one-way ANOVA with Tukey’s post hoc test. Data comparison between groups at different time points was performed using repeated-measures ANOVA with Bonferroni correction.