Fig. 6: RNA-seq and GSEA of TM- and 2-DG-preconditioned cancer cells.

A RNA-seq was performed on samples collected after preconditioning HA-low Has2^+Neo cells with 0.1 μg/ml TM or 0.1 mM 2-DG. RNA-seq data were compared with those from untreated cells (untreated control, UT). Heatmap of hierarchical clustering indicating DEGs between preconditioned and untreated cells (|log2FC | ≥ 2 and FDR < 0.1). Red and green indicate the upregulated and downregulated genes, respectively. B Number of DEGs between samples in each treatment group. The red and blue bars represent the number of upregulated and downregulated genes in each pair, respectively. C GSEA hallmark analysis of the pathways significantly upregulated in TM-preconditioned cells versus UT cells. D KEGG pathway analysis of RNA-seq data. Red and green indicate the upregulated and downregulated genes, respectively. E qRT-PCR analysis of Notch3 expression in Has2^ΔNeo and Has2^+Neo cells. Has2^+Neo cells were treated with 0.1 μg/ml TM or 0.1 mM 2-DG for 8 days and analyzed for Notch 3 expression by qRT‒PCR. The relative expression of Notch 3 mRNA was normalized to that of GAPDH. Data are the mean ± SD from n = 4. Two-tailed Student’s t-test. **p < 0.01. F Induction of cell surface Notch 3 expression by TM- and 2-DG preconditioning. Has2^+Neo cells were treated with 0.1 μg/ml TM or 0.1 mM 2-DG for 8 days and analyzed for cell surface Notch 3 expression by flow cytometry. Data are the mean ± SD from n = 3. Two-tailed Student’s t-test. **p < 0.01. G Suppression of mammosphere formation by Notch signaling inhibition. Has2^ΔNeo cells were cultured for 7 days in ultra-low attachment surface 24-well plates in spheroid-forming medium containing 50 µM LY411575, 50 µM LY3039478, 20 µM DAPT, or 1 µM LLNLe. The mammospheres were counted under a phase-contrast microscope. Scale bar: 100 µm. Data are the mean ± SD from n = 4. Two-tailed Student’s t-test. *p < 0.05, **p < 0.01. H Enhancement of cisplatin-induced apoptosis by inhibition of Notch signaling. Has2^ΔNeo cells were treated with 50 µM LY411575 for 8 days. Before staining with fluorescent Annexin V and PI, the cells were treated with 0-50 µM cisplatin for 16 h. Early and late apoptotic cells were represented as Annexin V⁺/PI^- and Annexin V⁺/PI⁺ subpopulations, respectively. Data are the mean ± SD from n = 4. Two-tailed Student’s t-test. *p < 0.05, **p < 0.01.