psat1  (Novus Biologicals)

Journal: Nature Communications

Article Title: Tumor metabolic adaptation induced by L-asparaginase reveals a vulnerability to PARP1/2 inhibitor in B-cell lymphomas

doi: 10.1038/s41467-026-70066-2

Figure Lengend Snippet: a L-[ 3 H(G)]-serine transport rate (cpm/10 6 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 13 C-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- 13 C]-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- 13 C]-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 13 C-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 table.

Article Snippet: Relative mRNA levels were obtained by real-time quantitative PCR (qPCR) on the StepOneTM Real-Time PCR System (Thermo Fisher Scientific) using the TaqMan assay primer set (Thermo Fisher Scientific) for murine Phgdh (Mm01623589_g1), Psat1 (Mm01613328_g1), Psph (Mm01197775_m1), Gls (Mm01257297_m1), Gclc (Mm00802658_m1), Gclm (Mm01324400_m1), Nqo1 (Mm01253561_m1), slc7a11 (Mm00442530_m1), slc6a9 (Mm00433662_m1), Asns (Mm01137310_g1), and the TaqMan Universal PCR Master Mix (4304437, Fisher Scientific) according to manufacturer’s instructions.

Techniques: Incubation, Quantitative Proteomics, Western Blot, Activity Assay, In Vivo, Derivative Assay

Journal: Nature Communications

Article Title: Tumor metabolic adaptation induced by L-asparaginase reveals a vulnerability to PARP1/2 inhibitor in B-cell lymphomas

doi: 10.1038/s41467-026-70066-2

Figure Lengend Snippet: a Percentage of PHGDH activity in Eµ- Myc (#688) cells treated with DMSO or indicated concentrations of the PHGDH inhibitor BI-4916 for 24 h in Asn-containing medium ( n = 3 independent experiments). b Percentage of dead Eµ- Myc (#688) cells (DAPI+) following 24 h of treatment with DMSO, ASNase (0.003 IU/ml) and/or BI-4916 (10 µM) in Asn-containing medium ( n = 4 independent experiments). c Four-day proliferation of Eµ- Myc (#688) cells treated as in ( b ) ( n = 5 independent experiments). d Total protein extracts prepared from Eµ- Myc (#506) cells stably expressing shRNA targeting the firefly luciferase (shSCR) or the murine Phgdh mRNA (two independent sh Phgdh #1 and #2) were immunoblotted for indicated proteins. e Relative quantification of immunoblots presented in ( d ). ( n = 4 independent experiments). f Percentage of dead Eµ- Myc (#506) cells (DAPI+) silenced (sh Phgdh #1 or #2) or not (shSCR) for Phgdh mRNA following 24 h incubation in Asn-containing medium supplemented (+) or not (−) with ASNase (0.003 IU/ml) ( n = 4 independent experiments). g Four-day proliferation of Eµ- Myc cells silenced or not (shSCR) for Phgdh mRNA, treated as in ( f ) ( n = 5 independent experiments). h Survival curves of WT C57BL/6 mice intravenously injected with Eµ- Myc (#506) cells stably expressing shRNA control (shSCR) or targeting the murine Phgdh mRNA (sh Phgdh #1), treated with Vehicle or ASNase every 48 h from day 7 until disease endpoint ( n = 10 mice/group). i Total protein extracts prepared from Eµ- Myc cells isolated from BCL of C57BL/6 mice presented in h , were immunoblotted for the indicated proteins (shSCR-Vehicle, n = 3; shSCR-ASNase, n = 3; sh Phgdh #1-Vehicle, n = 4; sh Phgdh #1-ASNase, n = 4 mice). V Vehicle, A ASNase. The samples derive from the same experiments but different gels for PHGDH, PSAT1, ERK2, and another for ASNS and ERK2 were processed in parallel. j Relative quantification of PHGDH protein levels presented in ( i ) Data are normalized to the control condition shSCR-Vehicle. Data are expressed as mean ± SD ( a , b , e , f , j ) or ± SEM ( c , g ). P -values are from one-way Anova followed by Tukey’s test ( a , e ), 2-way Anova followed by Tukey’s t -test ( b , f , j ), t -test ( c , g ), log-rank test ( h ), 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 table.

Article Snippet: Relative mRNA levels were obtained by real-time quantitative PCR (qPCR) on the StepOneTM Real-Time PCR System (Thermo Fisher Scientific) using the TaqMan assay primer set (Thermo Fisher Scientific) for murine Phgdh (Mm01623589_g1), Psat1 (Mm01613328_g1), Psph (Mm01197775_m1), Gls (Mm01257297_m1), Gclc (Mm00802658_m1), Gclm (Mm01324400_m1), Nqo1 (Mm01253561_m1), slc7a11 (Mm00442530_m1), slc6a9 (Mm00433662_m1), Asns (Mm01137310_g1), and the TaqMan Universal PCR Master Mix (4304437, Fisher Scientific) according to manufacturer’s instructions.

Techniques: Activity Assay, Stable Transfection, Expressing, shRNA, Luciferase, Quantitative Proteomics, Western Blot, Incubation, Injection, Control, Isolation

Journal: Nature Communications

Article Title: Tumor metabolic adaptation induced by L-asparaginase reveals a vulnerability to PARP1/2 inhibitor in B-cell lymphomas

doi: 10.1038/s41467-026-70066-2

Figure Lengend Snippet: a Schematic representation of the experimental design. Following 4 days of ASNase (0.003 IU/ml; 1 st challenge), wild-type (WT) Eµ- Myc (#506) cells were washed and re-seeded in Asn-containing medium (drug holiday). The resulting cell population (C1 cells) was treated with ASNase (2 nd challenge). b Percentage of dead (DAPI+) WT and C1 cells (from Eµ- Myc #506 cells) incubated in Asn-containing medium with or without (CTL) ASNase (0.003 IU/ml) for 24 h ( n = 4 independent experiments). c Proliferation of cells presented in ( b ) ( n = 4 independent experiments). d Total protein extracts prepared from Eµ- Myc (#506) cells treated as in ( a ) were immunoblotted for the indicated proteins. The samples derive from the same experiments, but different gels for PHGDH, another for PSAT1, another for PSPH, ASNS, ERK2, and another for ATF4 were processed in parallel. e Schematic representation of the two successive therapeutic challenges with Vehicle or ASNase in C57BL/6 mice bearing Eµ- Myc #506 cell-derived BCL. The 1 st therapeutic challenge resulted in V BCL and A BCL ( n = 6 mice/group). Malignant B cells from V BCL or A BCL were transferred into secondary recipient WT C57BL/6 mice. Seven days later, mice were treated with Vehicle or ASNase (2 nd therapeutic challenge), resulting in Vehicle- V BCL, Vehicle- A BCL, ASNase- V BCL, and ASNase- A BCL ( n = 6 mice/group). Survival curve of WT C57BL/6 mice intravenously injected with V BCL ( f ) or A BCL ( g ) malignant cells and treated with Vehicle or ASNase every 48 hours until disease endpoint ( n = 6 mice/group). h Principal-component analysis (PCA) of metabolites abundance (130 metabolites detected) in BCL harvested from Vehicle and ASNase-treated C57BL/6 mice bearing V BCL cells or A BCL cells ( n = 6 mice/group). i Heatmap representation of asparagine and serine relative abundance in BCL presented in ( e ) ( n = 6 mice/group). Malignant cells isolated from BCL of the Vehicle-treated mouse #738 and of the ASNase-treated mouse #750 were engrafted into secondary recipient WT C57BL/6 mice for the 2nd ASNase challenge. j Total protein extracts prepared from BCL described in ( i ) ( V BCL, n = 1; A BCL, n = 1; Vehicle A BCL, n = 6; ASNase- V BCL, n = 6), were immunoblotted for the indicated proteins. The samples derive from the same experiments, but different gels for PHGDH, another for PSAT1, PSPH, and another for ASNS and ERK2 were processed in parallel. Data are expressed as mean ± SD ( b , c ). P -values are from 2way Anova followed by Tukey’s test ( b ), t -test ( c .), log-rank test ( f , g ), and indicated as ns, not significant, **, p < 0.01; ***, p < 0.001 ****, p < 0.0001. For detailed individual P -value, please refer to the table.

Article Snippet: Relative mRNA levels were obtained by real-time quantitative PCR (qPCR) on the StepOneTM Real-Time PCR System (Thermo Fisher Scientific) using the TaqMan assay primer set (Thermo Fisher Scientific) for murine Phgdh (Mm01623589_g1), Psat1 (Mm01613328_g1), Psph (Mm01197775_m1), Gls (Mm01257297_m1), Gclc (Mm00802658_m1), Gclm (Mm01324400_m1), Nqo1 (Mm01253561_m1), slc7a11 (Mm00442530_m1), slc6a9 (Mm00433662_m1), Asns (Mm01137310_g1), and the TaqMan Universal PCR Master Mix (4304437, Fisher Scientific) according to manufacturer’s instructions.

Techniques: Incubation, Derivative Assay, Injection, Isolation