Fig. 5: Chaetocin-induced global transcriptome changes reveal the alteration of important hallmarks of cancer.

a Volcano plot of RNA sequencing data showing significantly (p < 0.05) up and down regulated genes by chaetocin (50 nM, 24 h) based on their log2 transformed expression data with false discovery rate (FDR) threshold of 0.05. b–c qPCR validation of top 4 upregulated (MLC1, ARL14EPL, HMOX1, CCDC64) (b) and downregulated (ANO8, ITGA2, ITGA11, TENM2) (c) genes obtained from RNAseq analysis. Data were normalized to untreated control. d Graph represents Gene Set Enrichment Analysis (GSEA) results pointing out chaetocin mediated positively and negatively enriched hallmark pathways based on their Normalized Enrichment Score (NES). e Representative enrichment plots for hallmark UV response-up, p53 and reactive oxygen species pathways. f Heatmaps of genes listed under UV response-up, p53 and ROS pathways from GSEA revealing significantly upregulated genes upon chaetocin treatment. Expression data were normalized to control condition and log 2 transformed (p < 0.05). g DCFDA flow cytometric analysis of ROS generation in U87MG cells treated with chaetocin in the presence or absence of N-acetyl-cysteine (NAC). h qPCR analysis showing that chaetocin treatment (100 nm, 24 h) upregulated TXNRD1, GCLM, and NQO1 gene levels in ROS-dependent manner. Data were normalized to no drug conditions. i qPCR analysis showing modulation of ARL14EPL and ANO8 gene levels by chaetocin treatment (100 nm, 24 h) in ROS-dependent manner. Data were normalized to Control conditions. ((*) and (***) denote P < 0.05 and P < 0.001, respectively, two-tailed Student’s t-test).