Fig. 5: ASNase-treated malignant B cells activate an antioxidant defense program that involves PHGDH activity to limit ROS.

a Relative ROS levels in Eµ-Myc (#688) cells cultivated for 15 h in Asn-free medium with (+) or without (–) Asn, ASNase (0.003 IU/ml) and N-acetyl-L-cysteine (NAC, 10 mM, 1 h) (n = 4 independent experiments). b GSSG/GSH ratio in Eµ-Myc (#688) cells cultivated in Asn-free medium, with (+) or without (−) Asn for 24 h (n = 4 biological replicates). c NRF2 and ATF4 expression levels in Eµ-Myc cells cultivated for 24 h as in (a). d Relative quantification of (c) (#506 cells, square, n = 2 independent experiments; #688 cells, triangle, n = 2 independent experiments). e Relative mRNA expression levels in Eµ-Myc (#688) cells cultivated as in (c) for 48 h (n = 3 independent experiments). f, g. Relative ROS levels in Eµ-Myc (#688) cells treated for 20 h in Asn-free medium with (+) or without (−) Asn (0.37 mM) and/or BI-4916 (10 µM) (f) or in Asn-containing medium with (+) or without ((−), DMSO), ASNase (0.003 IU/ml) and/or BI-4916 (10 µM) (g) (n = 4 independent experiments). h Percentage of dead Eµ-Myc (#506) cells (DAPI+) treated with (+) or without (−) DMSO, ASNase (0.003 IU/ml), BI-4916 (7.5 µM) and/or NAC (5 mM) for 48 h in Asn-containing medium (n = 4 independent experiments). i Heatmap representation of mRNA expression levels for the indicated genes, determined by RNAseq from Eµ-Myc cells isolated from 12 individual Eµ-Myc^Tg/+ mice. j As in (i) in BCL harvested from Eµ-Myc cells-bearing mice treated with Vehicle or ASNase (n = 1 /group). k slc7a11 mRNA expression levels (transcript per million) in selected tumor entities available on the TCGA database. l Total protein extracts from Eµ-Myc (#506) cells stably expressing control (CTL) or murine SLC7A11-encoding (SLC7A11 OE) vectors were immunoblotted for the indicated proteins. The samples derive from the same experiments but different gels for SC7A11, ERK2, and another for CD98 were processed in parallel. m Relative quantification of immunoblots presented in (l). Data normalized to SLC7A11 OE condition (n = 3 independent experiments). n Percentage of dead CTL and SLC7A11 OE-overexpressing Eµ-Myc (#506) cells (DAPI+) treated as in (h) (n = 5 independent experiments). Data are expressed as mean ± SD (a, d–h, m, n) or ± SEM (b). P-values are from 2-way Anova followed by Tukey’s test (a, f–h, n), t-test (b, m), one-way Anova followed by Tukey’s test (d, e), and indicated as ns, not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001 ****, p < 0.0001. For detailed individual P-value, please refer to the Source data table.