Fig 2: CPI-455 affected KDM5A and H3K4me3 expression in cochlear HCs. A, B The protein levels of KDM5A, KDM5B, KDM5C, and H3K4me3. GAPDH served as an internal control. Data are presented as mean ± SEM values. *P < 0.05, ***P < 0.001, ****P < 0.0001 compared with the control; ###P < 0.001, ####P < 0.0001 compared with CP only. The experiments were performed in triplicate. C Cochleae were stained for KDM5A (red) and Myosin 7a (green). D Cochleae were stained for H3K4me3 (red) and parvalbumin (green). Images were captured from the middle turns of the cochlea. Scale bars 10 µm

Fig 3: Interaction with p130 is essential for the recruitment of RBP2 onto the E2F-responsive promoters. (A) Mutation of LxCxE motif in RBP2 leads to loss of binding of RBP2 to the E2F-responsive promoters. Chromatin affinity purification (ChAP) assay was performed in HeLa cells stably expressing either wild type (wt.) SFB-RBP2, SFB-RBP2 (E1377K), SFB-RBP2 (H483G, E485Q) or SFB-RBP2 (W1625A) are shown. The amount of RBP2 pulled down in each sample is shown in Supplementary Figure S5A. The error bars represent S.D. Tukey's multiple comparison test *P=0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: not significant, P>0.05. (B) ChIP assay was performed in cells expressing either RBP2 wt., RBP2 E1377K or empty SFB-pcDNA5-FRT vector. The H3K4me3 levels on different promoters are shown. H3 levels are shown in Supplementary Figure S6B. The error bars represent S.D. Tukey's multiple comparison test was employed to calculate the significance *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: not significant, P > 0.05. (C) RBP2 affects transcription of E2F-responsive gene. G1/S synchronized cells were treated with either siRNA against 3'UTR region of RBP2 or control (luciferase) siRNA in cells stably expressing either empty vector, RBP2 wt., RBP2 E1377K or RBP2 H483G, E485Q. cDNA synthesized from total RNA was amplified using real-time PCR for indicated mRNAs. All signals were normalized to actin mRNA levels from respective samples. The expression level is relative to control siRNA-treated cells (which is arbitrarily set to 1) from respective cell line but for ease of understanding only one control bar is shown. The error bars represent S.D. Dunnett's multiple comparison test was employed to calculate the significance*P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: not significant, P > 0.05.

Fig 4: The transcriptome of KDM5A-KO cells.A Hierarchical clustering of gene expression profiles from 3 biologically independent samples based on Pearson correlation coefficient in WT and KDM5A-KO cells. Colors from green to red indicate weak-to- strong correlation. WT: wild type, 143B cell line. B Volcano plot of all genes analyzed in the present study. Blue, significantly differently downregulation genes. Red, significantly differently overregulation genes. Black, nonsignificantly differently expressed genes (DEG). C, D GO analysis of upregulated (C) and downregulated genes (D) for biological processes in KDM5A-KO cells. E The genes that were involved in >4 pathways of enrichment sets, including IL6 (shared by eight pathways), TNFAIP3 (shared by six pathways), IL15 and INHBA (shared by five pathways), CCL2, ICMA1, IL1B, IL7, IRF7, ITGB3, LIF, and MMP14 (shared by four pathways). All core enrichment genes in the 13 enriched sets were shown in Supplementary Table S1. F A protein–protein interaction (PPI) network of the 12 enrichment genes was constructed by using the STRING online database (https://string-db.org/). G The expression of the enrichment genes in KDM5A-KO cells was detected using RT-qPCR analyses. Data were shown as the mean ± S.D (n = 3). *p < 0.05, **p < 0.01, ***p < 0.01.

Fig 5: Downregulation of KDM5A reduced CP-induced apoptosis in HEI-OC1 cells. A–C Effects of KDM5A on early (Annexin V-FITC positive, PI negative) and late (Annexin V-FITC/PI double positive) cell apoptosis were (A) determined via flow cytometry and (B, C) quantified. The data are presented as three independent experiments' mean ± SEM values. ****P < 0.0001 compared with the control; ####P < 0.0001 compared with CP only. D–F Representative images and histogram analysis of CellROX™ green in HEI-OC1 cells. Scale bar = 10 µm. The data are presented as three independent experiments' mean ± SEM values. ***P < 0.001, ****P < 0.0001 compared with the control; ####P < 0.0001 compared with CP only. G–I Representative images and histogram analysis of Rhodamine 123 in HEI-OC1 cells. Scale bar 10 µm. The data are presented as three independent experiments' mean ± SEM values. ****P < 0.0001 compared with the control; ###P < 0.001 compared with CP only. J–N OCRs and bioenergetics parameters in HEI-OC1 cells from each group. The data are presented as mean ± SEM values. **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control; ##P < 0.01, ####P < 0.0001 compared with CP only. The experiments were performed in triplicate