Fig. 2: De novo serine biosynthesis increased in B-cell lymphomas treated with ASNase in vivo.

a L-[³H(G)]-serine transport rate (cpm/10⁶ cells) in Eµ-Myc (#688) cells following 24 h incubation in serine/glycine-containing medium, with (+) or without (−) Asn (0.37 mM) and ASNase (0.003 IU/ml) (left, 15 min uptake, n = 4 independent experiments; right, 3 h uptake, n = 3 independent experiment). Relative abundance (peak area) of total intracellular serine (b) and glycine (c) levels in Eµ-Myc (#688) cells cultivated for 24 h in Asn/serine/glycine-free medium, supplemented (+) or not (−) with serine, glycine (Ser/Gly; 0.4 mM/0.4 mM), Asn (0.37 mM) and ASNase (0.003 IU/ml) (n = 3 biological replicates). d Total protein extracts prepared from live Eµ-Myc #506 (left) and #688 (right) cells cultivated for 24 h in glutamine (Gln) and Asn-free medium supplemented (+) or not (−) with Gln (2 mM), Asn (0.37 mM) or ASNase (0.003 IU/ml), were immunoblotted for the indicated proteins. The samples derive from the same experiments, but different gels for PHGDH, PSPH, ERK2, another for PSAT1, ASNS, another for ATF4, another for GLUL, and another for GOT1 were processed in parallel. e Relative quantification of immunoblots presented in (d). (#506 cells, square, n = 2 independent experiments; #688 cells, triangle, n = 2 independent experiments). Relative abundance (peak area) of ¹³C-labelled serine M + 3 (f) and glycine M + 2 (g) isotopologues in Eµ-Myc (#688) cells cultivated for 24 h in glucose and asparagine-free medium supplemented with 25 mM [U-¹³C]-glucose and with (+) or without (−) Asn (0.37 mM) and ASNase (0.003 IU/ml) (n = 4 biological and two technical replicates). h Total protein extracts prepared from BCL harvested in Eµ-Myc (#506) cells-bearing C57BL/6 mice, treated with Vehicle or ASNase every 48 h till disease endpoint, were immunoblotted for the indicated proteins (Vehicle, n = 4 mice; ASNase, n = 10 mice). The samples derive from the same experiments, but different gels for PHGDH, PSAT1, ERK2, another for PSPH, another for ASNS, and another for ATF4 were processed in parallel. i Percentage of PHGDH activity in BCL harvested in Eµ-Myc cells-bearing C57BL/6 mice treated as in (h) (n = 9 mice/group). j Schematic representation of in vivo [U-¹³C]-glucose consecutive bolus protocol performed in Vehicle and ASNase-treated C57BL/6 mice bearing OxPhos-dependent Eµ-Myc #688 cell-derived BCL. k Relative abundance (peak area) of ¹³C-labelled serine (M + 3) and glycine (M + 2) isotopologues in BCL of mice presented in (j) (n = 4 mice/group). For all graphs, data are expressed as mean ± SD. P-values are from one-way Anova followed by Tukey’s test (a, f, g), or followed by Dunnett’s test (e), 2way Anova followed by Tukey’s t-test (b, c), t-test (i., k.) 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.