Fig. 2: NOTCH1 activation affects the response to OxPhos-inhibition of primary pre-LSCs.

a Notch1 gene mutation in SCL^tgLMO1^tg preleukemic and leukemic thymocytes. b Overexpression of NOTCH1 protein by flow cytometry. Thymocytes from wt- and NOTCH1^tg mice were stained with anti-NOTCH1 antibody or an isotype control. c dose–response of primary pre-LSCs to IACS-010759, co-cultured on MS5-DL4 stromal cells followed by 48 h drug treatment (mean ± SD, n = 3 independent experiments in triplicates). d Dose–response of pre-LSCs to IACS-010759 treatment, co-cultured on MS5 stromal cells followed by 48 h drug treatment (mean ± SD, n = 3.independent experiments in triplicates). e Effects of IACS-010759 treatment (133 nM or DMSO) on viability of pre-LSCs cultured on MS5-DL4 (mean ± SD, n = 3 independent experiments). f Effects of IACS-010759 treatment (133 nM or DMSO) on viability of pre-LSCs cultured on MS5 (mean ± SD, n = 3 independent experiments). g Pre-LSC sensitivity to IACS-010759 for the indicated genotypes. The Area under the curves (AUC) were computed from dose–response data illustrated in (c). Shown is the AUC difference obtained between DMSO (black ring) and drug-treated cells (p < 0.00001, multiple unpaired t tests, n = 3 mice per genotype, in triplicates); with (green) and without DL4 (blue), in pre-LSCs with or without NOTCH1 oncogene in the absence of DL4. h Basal oxygen consumption rate (OCR) in T-lymphocytes (n = 7), NOTCH1-wt (n = 3) and -mutant cell lines (n = 7) by Mito Stress Test assay (mean ± SD, n = 3 independent measurements for each line, 4 replicates); one-way ANOVA; *p < 0.05; ***p < 0.001. i IC₅₀ for basal OCR inhibition (Supplementary Figs. 3c and 4) for T-lymphocytes (n = 4) and NOTCH1-wt (n = 3) and -mutated T-ALL cell lines (n = 7) (mean ± SD, n = 3 independent experiments for each line, 4 replicates); one-way ANOVA; *p < 0.05; ***p < 0.001. j IC₅₀ for viability inhibition for T-lymphocytes (n = 5), NOTCH1-wt (n = 3) and -mutant cell lines (n = 7) treated with IACS-010759 (0–123 nM, 96 h); (mean ± SD, n = 3 independent experiments); one-way ANOVA; ns-no significance, **p < 0.005. k Viable cell number in T-lymphocytes (n = 7), NOTCH1-wt (n = 3) and -mutant (n = 7) T-ALL cell lines, treated with IACS-010759 (1, 10, 100 nM, 96 h), (mean ± SD, n = 3 independent experiments per line, 3 replicates); two-way ANOVA: ns-no significance, *p < 0.05; **p < 0.005; ***p < 0.001; and ****p < 0.0001. l ROS (MFI) treated with IACS-010759 (1, 10, 100 nM; 96 h) in T-lymphocytes (n = 6), T-ALL cell lines with wt- (n = 3) and mutant NOTCH1 (n = 7); (mean ± SD, n = 3 independent experiments per line, 3 replicates); two-way ANOVA: ns-no significance, *p < 0.05; **p < 0.005; ***p < 0.001; and ****p < 0.0001.