Fig. 2

Enrichment analysis of TEPA-treated cells a and b, GSEA enrichment plot of EMT-related genes identified by RNA-Seq analysis in MDA-MB-231 cells treated with TEPA for 8 (left panel) and 24 (right panel) hours. Downregulation of the MSigDB Hallmark EMT gene set is consistently observed at both time-points. a’ and b’, Heatmap showing EMT gene set leading-edge genes at MDA-MB-231 cells treated with TEPA for 8 (a’) and 24 (b’) hours. Heatmap color intensity is proportional to row-scaled VST-normalized expression.c, d, GSEA enrichment plot showing downregulation of TGFB1 early responsive genes identified by RNA-Seq analysis in MDA-MB-231 cells treated with TEPA for 8 (left panel) and 24 (right panel) hours. c’, d’, Heatmap showing MSigDB’s VERRECCHIA EARLY RESPONSE TO TGFB1 gene set leading-edge genes at MDA-MB-231 cells treated with TEPA for 8 (c’) and 24 (d’) hours. The heatmap color intensity is proportional to row-scaled VST-normalized expression. e Bar-plot showing common negatively enriched gene terms in MDA-MB-231 cells treated with TEPA for 8 and 24 h and SH-SY5Y treated with TEPA for 24 h. Enrichment analysis was performed with the enrich R package. A consistent downregulation of EMT and cholesterol metabolism can be observed in both breast cancer and neuroblastoma cells. Furthermore, downregulation of several signaling pathways (IL-2/STAT5, mTORC1 and KRAS) is observed in both cell models following TEPA treatment. f Up, GSEA enrichment plot of EMT-related genes identified by RNA-Seq analysis in DIPG010 cells treated with TEPA for 24 h. Downregulation of the MSigDB Hallmark EMT gene set is consistently observed at this time-point. f’, Heatmap showing EMT gene set leading-edge genes at DIPG010 cells treated with TEPA for 24 h. Heatmap color intensity is proportional to row-scaled VST-normalized expression. g Up, GSEA enrichment plot showing downregulation of early TGFB1 early responsive genes identified by RNA-Seq analysis in DIPG010 cells treated with TEPA for 24 h. g’, Heatmap showing MSigDB’s VERRECCHIA EARLY RESPONSE TO TGFB1 gene set leading-edge genes at DIPG010 cells treated with TEPA. The heatmap color intensity is proportional to row-scaled VST-normalized expression. h Analysis of TGF-β/SMAD2/3 and TGF-β/AKT/mTOR signaling pathways at protein levels. Western blot analysis of SMAD2, phospho-SMAD2, AKT, phospho-AKT, ERK1/2, and phosphor-ERK1/2 following treatment of MDA-MB-231, SH-SY5Y, BE2C, DIPG007, and DIPG010 cells with TEPA (0, 4 and 8 mM for MDA-MB-231 and DIPG010, and 0 and 2 mM for SH-SY5Y and DIPG007) for 24 h. GAPDH has been used as a positive and an endogenous control for normalization