Fig. 5

SP2509/CFZ treatment affects cell cycle regulation and apoptosis. A Volcano plot showing differentially expressed genes (DEGs) with |Log2FoldChange|> 0.5 between CFZ/SP2509 treatment and DMSO control. Analysis was performed 24 h post-treatment. Blue dots represent significantly downregulated genes related to cell cycle processes, orange dots represent significantly downregulated genes belonging to the replication-dependent histones family. B Dot plot graph of enriched Gene Ontology (GO) terms from genes in A. The nine GO processes with the largest gene ratios are plotted in order of fold enrichment. The size of the dots represents the number of genes in the significant DEG list associated with the GO term, and the color of the dots represents the P-adjusted values. C Representative heatmap of RT-qPCR validation performed in the U266 cell line treated with CFZ (2.5 nM), SP2509 (1 µM), or the combination for 48 h. HUPO was used as a housekeeping gene. DMSO-treated cells were used to normalize mRNA expression levels equal to 1. D Heatmap showing non-supervised hierarchical clustering of normalized reverse phase protein array (RPPA) intensities using the average-linkage method and Pearson distance. U266 cells were treated with DMSO (n = 3), CFZ (2.5 nM, n = 3), SP2509 (1 µM, n = 3), CFZ/SP2509 (n = 3), GSK-LSD1 (1 µM, n = 2), or CFZ/GSK-LSD1 (n = 2). Pellets were collected 48 hpt. E Western blot analysis of U266 cells treated with the indicated drugs. Pellets were collected 48 h post-treatment. Vinculin was used for protein loading normalization. DEG: differential expressed genes, GO: gene ontology; RPPA: reverse phase protein array