Fig. 4: Inhibition of KMT9 impairs tumour cell proliferation.
a, b CETSA for KMT9 in PC-3M cells treated with vehicle (DMSO), 1 µM KMI169 or KMI169Ctrl. Representative Western blots (a) and quantification (b) showing increased melting temperatures (ΔTm) of endogenous KMT9 upon treatment with KMI169 compared to KMI169Ctrl or DMSO. Data represent means ±s.d (n = 3 biologically independent experiments). c Levels of H4K12me1 in PC-3M prostate tumour cells cultured in the presence of DMSO (-), 500 nM KMI169 or 500 nM KMI169Ctrl for one (D1), two (D2) or three (D3) days were analysed by Western blot using the indicated antibodies. Histone H4 was used as control. d Concentration of half-maximal growth inhibition (GI50) determined by MTT cell viability assays for KMI169 and KMI169Ctrl in PC-3M cells. Data represent means ± s.d (n = 3 biologically independent samples). e Table summarizing GI50 values for KMI169- and KMI169Ctrl-mediated inhibition of proliferation of different tumour cells determined by MTT assays. Data represent means ± s.d (n = 3 biologically independent samples). f Diagram showing the number of differentially expressed genes (DEGs) in PC-3M cells treated four days with 360 nM KMI169 (+) or DMSO (-). g Venn diagram showing the intersection of DEGs upon treatment with KMI169 or RNAi-mediated knockdown of KMT9α. A one-sided hypergeometric test was used to calculate the significance of the overlaps (p = 5.6 × 10-144, r = 2.0, r: representation factor) h Venn diagram showing the intersection of DEGs upon treatment of PC-3M cells with KMI169 and KMT9α/β gene occupancy determined by ChIP-seq. A one-sided hypergeometric test was used to calculate the significance of the overlaps (p = 4.924 × 10-5, r = 1.2). i Enriched pathway analysis for 382 genes differentially expressed upon KMI169 treatment and showing KMT9 gene occupancy. Bars represent individual Benjamini p-values derived from GO enrichment analysis. j Heatmap displaying mRNA levels of differentially expressed genes in PC-3M cells cultured in the presence of DMSO (-) or 360 nM KMI169. FC: fold change. k QRT-PCR analysis showing relative mRNA levels of the indicated genes in PC-3M cells cultured in the presence of DMSO or 360 nM KMI169. Data represent means +s.d. MYB (n = 4, p = 0.0003), AURKB (n = 4, p = 0.0005), FOXA2 (n = 4, p = 0.004), CDK2 (n = 4, p = 0.0053), BIRC5 (n = 3, p = 0.0161), E2F1 (n = 3, p = 0.0058), E2F8 (n = 4, p = 0.0024), CDC6 (n = 3, p = 0.002), LIG1 (n = 4, p = 0.0014). (n represents the number of biologically independent samples. *p < 0.05, **p < 0.005, ***p < 0.001 by two-tailed Student’s test.
