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

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 Total protein extracts prepared from Eµ- Myc #688 cells cultured in Asn-free medium, supplemented (+) or not (−) with Asn (0.37 mM) and ASNase (0.003 IU/ml) for 24 h, were immunoblotted for the indicated proteins. Etoposide treatment (1 µg/ml) for 3 h was used as a positive control. b Relative quantification of immunoblots presented in ( a ). (#506 cells, square, n = 2 independent experiments; #688 cells, triangle, n = 3 independent experiments). c As in a for the indicated proteins expressed in Eµ- Myc #506 and #688 cells. From #506 cells, the samples derive from the same experiments, but different gels for P-ATR (S428), P-Chk1 (S345), KAP1, ERK2, another for ATR, another for Chk1, P-RPA32 (S33), P-KAP1 (S824), another for RPA32, and another for γH2AX were processed in parallel. From #688 cells, the sample derived from the same experiments, but different gels for P-ATR (S428), P-Chk1 (S345), P-KAP1 (S824), ERK2, another for ATR, Chk1, KAP1, another for P-RPA32 (S33), another for RPA32, and another for γH2AX were processed in parallel. d As in ( b ) for immunoblots presented in ( c ) (#506 cells, square, n = 2 independent experiments; #688 cells, triangle, n = 3 independent experiments). e γH2AX expression levels in Eµ- Myc cells (#506 and #688) following 24 h incubation in Asn-containing medium supplemented (+) or not (−) with ASNase (0.003 IU/ml) and/or N-acetyl-L-cysteine (NAC, 10 mM). f Relative quantification of γH2AX expression in Eµ- Myc cells presented in ( e ) (#688 cells, triangle, n = 2 independent experiments; #506 cells, square, n = 1). g γH2AX expression levels in Eµ- Myc (#688) cells treated (+) or not (−) for 24 h with DMSO (−/−), ASNase (0.003 IU/ml) and/or BI-4916 (10 µM). h Relative quantification of γH2AX expression levels in Eµ- Myc cells presented in ( g ). (#688 cells, triangle, n = 2 independent experiments; #506 cells, square, n = 1). i Total protein extracts prepared from BCL harvested from C57BL/6 mice engrafted with control (CTL) or V5-tagged murine PHGDH-overexpressing (PHGDH OE) Eµ- Myc (#506) cells and treated with Vehicle or ASNase from day 7 until disease endpoint, were immunoblotted for the indicated proteins (CTL-Vehicle, n = 3; CTL-ASNase, n = 3; PHGDH OE-Vehicle, n = 4; PHGDH OE-ASNase, n = 4 mice). The samples derive from the same experiments, but different gels for γH2AX, ERK2 (upper), another for P-ChK1 (S345), ERK2 (middle), another for Chk1, another for PHGDH, ERK2 (lower), and another for V5 were processed in parallel. j Relative quantification of γH2AX and P-Chk1 (Ser345) expression levels in BCL presented in ( i ). V Vehicle, A ASNase. For all graphs, data are expressed as mean ± SD. P -values are from one-way Anova followed by Tukey’s test ( b , d ), 2-way Anova followed by Tukey’s test ( f , h , j ) 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: Cell Culture, Positive Control, Quantitative Proteomics, Western Blot, Derivative Assay, Expressing, Incubation, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PHGDH drives 5-FU chemoresistance in colorectal cancer through the Hedgehog signaling

doi: 10.1186/s13046-025-03447-y

Figure Lengend Snippet: PHGDH expression is heterogeneous in CRC tissues and in PDCOs. A Representative H&E and PHGDH staining of PDCOs by IHC detection (left, high PHGDH; right, low PHGDH) (magnification 20 ×, scale bar: 100 μm). B PHGDH protein expression levels in PDCOs (western blot and relative bargraph, showing quantification of PHGDH protein levels). Vinculin was used as loading control. The dashed line represents an arbitrary threshold used to cluster the organoids into high and low PHGDH. Western blot image is representative of at least three independent experiments. C PHGDH protein expression levels in healthy and tumor tissues from CRC patients’ specimens. Western blot of representative samples within the cohort. Vinculin was used as loading control. D Box and Whisker plot comparing the expression distribution of PHGDH in healthy ( n = 19) and tumor ( n = 24) CRC tissues. Values of variance of the two datasets are reported at the top of each box (healthy, s 2 = 0.0638; tumor, s 2 = 0.2524). Levene’s test for equality of variance resulted in a f-ratio value of 14.86044 and a p -value of 0.0004, indicating that the variances of the two populations are significantly different from each other. E Representative images of H&E and low (left) and high (right) PHGDH IHC staining on human-derived tumors (magnification 10 ×, scale bar: 200 μm), along with the corresponding quantification (PHGDH H-score) across 14 different tumor samples ( F ). Tumors are categorized into low and high PHGDH expression groups based on H-score values. t-test **** p < 0.0001

Article Snippet: HCT8 and LS174T cells were silenced with PHGDH human shRNA plasmid (Origene) or scrambled shRNA (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Expressing, Staining, Western Blot, Control, Whisker Assay, Immunohistochemistry, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PHGDH drives 5-FU chemoresistance in colorectal cancer through the Hedgehog signaling

doi: 10.1186/s13046-025-03447-y

Figure Lengend Snippet: PHGDH expression correlates with resistance to 5-FU in CRC patients and PDCOs. A Retrospective analysis of 50 CRC patients showing the correlation between PHGDH expression level and therapy response (R: Responder, NR: Non Responder). B Violin plots show PHGDH gene expression profile in normal colon (Normal), primary (Tumor) and metastases (Metastases) of colon cancer obtained by online analysis platform on TNMplot. C Kaplan–Meier plotter of high and low PHGDH expression in CRC. OS and RFS in CRC patients expressing high PHGDH (red) and low PHGDH (black) levels. D PDCOs response to 5 μM 5-FU treatment. Cell viability was assayed after 7 days of treatment by CellTiter-Glo 3D reagent

Article Snippet: HCT8 and LS174T cells were silenced with PHGDH human shRNA plasmid (Origene) or scrambled shRNA (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Expressing, Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PHGDH drives 5-FU chemoresistance in colorectal cancer through the Hedgehog signaling

doi: 10.1186/s13046-025-03447-y

Figure Lengend Snippet: PHGDH supports resistance to 5-FU. A PHGDH protein levels in HCT8 and RKO cells following shRNA silencing or overexpression, respectively. Vinculin immunoblot was performed to ensure equal loading. B-C Proliferation rate of HCT8 and RKO cells after stable transfection. Data are reported as mean ± SEM from three independent experiments. D-E Survival fraction of HCT8, HCT8 shPHGDH, RKO and RKO OE PHGDH treated with increasing concentration of 5-FU for 48 h. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. F γH2AX levels in transfected and parental HCT8 and RKO cells. H2AX and vinculin were used as controls. Each image is representative of at least three independent experiments. G Colony formation assay of HCT8, HCT8 shPHGDH, RKO and RKO OE PHGDH cells grown either in the absence or presence of 5 μM 5-FU. After 48 h, cells were detached, counted, and 1000 cells were plated in new dishes, and incubated for at least 10 days. Formed colonies were stained with Crystal Violet. Representative images are shown. Data are reported as mean ± SEM from three independent experiments; t-test; ** p < 0.01, **** p < 0.0001

Article Snippet: HCT8 and LS174T cells were silenced with PHGDH human shRNA plasmid (Origene) or scrambled shRNA (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: shRNA, Over Expression, Western Blot, Stable Transfection, Concentration Assay, Transfection, Colony Assay, Incubation, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PHGDH drives 5-FU chemoresistance in colorectal cancer through the Hedgehog signaling

doi: 10.1186/s13046-025-03447-y

Figure Lengend Snippet: PHGDH confers aggressiveness to CRC cells. A Invasive abilities of PHGDH-silenced or PHGDH-overexpressing cells. CRC cells were seeded in the upper compartment of 8 μm Transwell system coated with Matrigel. After 16 h invaded cells were stained with Crystal Violet and counted. Representative images of invaded CRC cells are shown below the bargraphs (magnification 20 ×, scale bar: 100 μm). Data are reported as mean ± SEM from three independent experiments; t-test; **** p < 0.0001. B PHGDH silenced or overexpressing cells were treated with 5 μM 5-FU for 48 h then P1 spheres forming ability was performed seeding 700 cells/well in 96-well plate. After 7 days colonspheres were counted, and volumes were calculated as described in Methods. Representative images of CRC spheroids are shown below the bargraphs (magnification 20 ×, scale bar: 100 μm). Data are reported as mean ± SEM from three independent experiments; bargraph represents the fold increase relative to respective CTRL; t-test; *** p < 0.001, **** p < 0.0001. C Cells disaggregated from P1 spheres were re-plated to test secondary spheroid (P2) formation ability in HCT8, HCT8 shPHGDH cells, RKO and RKO OE PHGDH. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01. D mRNA expression levels of genes involved in CSC phenotype acquisition and EMT program in P1 colonspheres. Data are reported as mean ± SEM from three independent experiments; t-test; ** p < 0.01, *** p < 0.001, **** p < 0.0001. E Membrane EpCAM, CD24, CD133 and CD44 expression levels in CRC cells evaluated in P1 colonspheres by flow cytometry. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: HCT8 and LS174T cells were silenced with PHGDH human shRNA plasmid (Origene) or scrambled shRNA (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Staining, Expressing, Membrane, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PHGDH drives 5-FU chemoresistance in colorectal cancer through the Hedgehog signaling

doi: 10.1186/s13046-025-03447-y

Figure Lengend Snippet: PHGDH modulation affects Hedgehog signaling pathway. A PHGDH protein level in cytosolic or nuclear compartment in HCT8 cells treated with 5 μM 5FU for 48 h. HSP90 and H3 were used as loading control for cytosolic and nuclear compartment, respectively. B Co-immunoprecipitation of PHGDH and c-Myc in shCTRL or shPHGDH cells. An anti-PHGDH antibody was used to pull down PHGDH, then western blotting for c-Myc was performed. C PCA displays the distribution of samples based on their gene expression profiles, projected onto the first two principal components (PC1 and PC2). PC1 (x-axis) captures 92% of the total variance, while PC2 (y-axis) captures 3%. Samples are color-coded according to their group: black points indicate shCTRL replicates, red and brown points indicate shPHGDH replicates. D Pathway Enrichment Analysis of Differentially Expressed Genes illustrating the top enriched pathways in the comparison between shCTRL and shPHGDH samples. The analysis was performed using the MSigDB Hallmark gene set. The x-axis represents the Normalized Enrichment Score (NES) , which indicates the up/down-regulation degree of the altered pathways: Pathways with positive NES values are up-regulated in shPHGDH samples compared to shCTRL, while pathways with negative NES values down-regulated. The associated adjusted p -values (Benjamini-Hochberg) are displayed to the right of each bar. E GSEA focused on the Hallmark HH Signaling pathway. The plot displays the resulting normalized enrichment score (NES, green line) of the ranked list of DEGs across the dataset. The leading edge, i.e. the genes contributing most significantly to the enrichment, are displayed below each bar which represents the position of the gene in the ranked list. Each gene is also annotated by the log2FC (boxes above the bars), providing an indication of its expression change in the comparison. The red dashed lines indicate the maximum and minimum NES achieved. F Heatmap of HH Pathway Target Genes: displays the expression levels of HH pathway target genes across different samples. The genes are organized based on their involvement in specific cellular processes annotated on the top. Sample clustering is shown via a dendrogram at the top of the heatmap. G mRNA expression levels of genes involved in HH pathway analyzed by qPCR. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. H-I mRNA expression levels of PTCH1, GLI1 and GLI2 in PHGDH-silenced cells or PHGDH-overexpressing and PDCOs analyzed by qPCR using parental cells or high PHGDH organoids as comparator. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01,*** p < 0.001, **** p < 0.0001

Article Snippet: HCT8 and LS174T cells were silenced with PHGDH human shRNA plasmid (Origene) or scrambled shRNA (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Control, Immunoprecipitation, Western Blot, Gene Expression, Comparison, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PHGDH drives 5-FU chemoresistance in colorectal cancer through the Hedgehog signaling

doi: 10.1186/s13046-025-03447-y

Figure Lengend Snippet: GLI1 inhibition resensitizes CRC cells to 5-FU treatment. A GLI1 protein level in shCTRL or shPHGDH cells. β-Actin immunoblot was performed to ensure equal loading. B Representative images of western blot for GLI1, PHGDH and β-Actin of HCT8 cells silenced with GLI1 shRNA. C Immunoblot of PHGDH in HCT8 shCTRL or shPHGDH cells treated with 2 μM JC19 for 48 h. β-Actin immunoblot was performed to ensure equal loading. D Effects of 5-FU on GLI1-silenced HCT8 cells. Cells were treated for 48 h with increasing concentration of 5-FU. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01, **** p < 0.0001. E–F Survival fraction of transfected cells (E) and PDCOs (F) treated or not with 5 μM 5-FU in combination with 2 μM JC19 for 48 h (CRC cells) or 7 days (PDCOs). Data are reported as mean ± SEM from three independent experiments; ANOVA; * p < 0.05, *** p < 0.001, **** p < 0.0001. ϕ indicates synergistic effect (Bliss test > 0) with respect to single treatments. G PTCH1, GLI1 and GLI2 mRNA expression level in HCT116 and HCT116R cells. HCT116 was used as comparator. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01. H mRNA expression levels of HH pathway target genes analyzed by qPCR. HCT116 was used as comparator. Data are reported as mean ± SEM from three independent experiments; t-test; * p < 0.05, ** p < 0.01. I Survival fraction of HCT116 and HCT116R cells treated or not with 5 μM 5-FU in combination with 2,5 μM JC19 for 48 h. Data are reported as mean ± SEM from three independent experiments; ANOVA; *** p < 0.001, **** p < 0.0001. ϕ indicates synergistic effect (Bliss test > 0) with respect to single treatments. J GSEA of HH in R vs NR colon cancer patients from publicly available TCGA datasets (TCGA-READ, TCGA-COAD)

Article Snippet: HCT8 and LS174T cells were silenced with PHGDH human shRNA plasmid (Origene) or scrambled shRNA (Origene) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Inhibition, Western Blot, shRNA, Concentration Assay, Transfection, Expressing

Journal: Cell Metabolism

Article Title: Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation

doi: 10.1016/j.cmet.2019.06.003

Figure Lengend Snippet:

Article Snippet: Rabbit anti-PHGDH , Bethyl Laboratories , Cat#A304-732A RRID: AB_2620927.

Techniques: Virus, Recombinant, Polymer, Protease Inhibitor, Membrane, Adhesive, Cell Viability Assay, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Modification, Software, Quantitative Proteomics, Sterility

Journal: Oncotarget

Article Title: Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth.

doi: 10.18632/oncotarget.6426

Figure Lengend Snippet: Figure 3: Gln deprivation promotes the serine pathway. (A) Representative gel resulting from a 2D-DIGE experiment comparing the proteome of HL-60 cultured in the absence of Gln or Glc; proteins overexpressed in the absence of Gln indicated by an arrow (left) are listed (right) with indication of the fold-change (P < 0.05). (B) Scheme of the serine pathway from 3-phosphoglycerate (3PG). Representative immunoblots for (C) PHGDH and (D) PSAT in HL-60 leukemia cells incubated for 48 hours in medium containing the indicated decreasing concentrations of Gln. (E) Representative immunoblots for PHGDH in HL-60 leukemia cells exposed for 48 hours (in medium containing Glc and/or Gln) to the indicated increasing concentrations of L-asparaginase. Representative immunoblots for (F) ATF4 and (G) phospho- eIF2 after 6 hours Gln withdrawal in HL-60 leukemia cells. The immunoblot experiments were repeated 2–3 times with similar results.

Article Snippet: PHGDH siRNA were from Thermo Scientific Dharmacon (ON-TARGET plus Human PHGDH, smart pool) and PHGDH shRNA was from Origene.

Techniques: Cell Culture, Western Blot, Incubation

Journal: Oncotarget

Article Title: Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth.

doi: 10.18632/oncotarget.6426

Figure Lengend Snippet: Figure 4: Both exogenous serine and PHGDH expression support leukemia cell growth. (A) Bar graph represents the growth (%) of HL-60 leukemia cells transfected with either control or PHGDH siRNA and incubated for 48 hours in a medium with or without serine (n = 3). (B) Representative immunoblots for PHGDH and phospho-S6RP in the corresponding culture conditions. Bar graphs represent the growth (%) of HL-60 leukemia cells (C) incubated in a serine-free (or serine-containing) medium (n = 3) or (E) transfected with a PHGDH shRNA (or a control shRNA), and incubated for 48 hours in a medium with or without Gln (n = 2). (D) Representative immunoblots of phospho-S6RP in the different conditions described in C; the immunoblot experiments were repeated 2–3 times with similar results. (F) Effect of Gln replenishment on HL-60 cell growth after 72 hours Gln withdrawal and transfection with either PHGDH or control siRNA (n = 6).

Article Snippet: PHGDH siRNA were from Thermo Scientific Dharmacon (ON-TARGET plus Human PHGDH, smart pool) and PHGDH shRNA was from Origene.

Techniques: Expressing, Transfection, Control, Incubation, Western Blot, shRNA

Journal: Oncotarget

Article Title: Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth.

doi: 10.18632/oncotarget.6426

Figure Lengend Snippet: Figure 5: PHGDH expression allows to reduce the extent of oxidative stress induced upon Gln withdrawal. Bar graphs represent the amounts of total glutathione in HL-60 leukemia cells exposed to a medium (A) with or without Gln (for 6 hours) and (B) with or without exogenous serine (for 48 hours) (n = 3). (C) ROS production (as determined using the H2- DCFDA probe) in HL-60 leukemia cells exposed for 6 hours to a medium with or without Gln (n = 4). Effects of H2O2 (10 µM, 48 hours) on (D) the expression of PHGDH in HL-60 cells and (E) on the growth of HL-60 cells transfected with control or PHGDH shRNA and treated with L-asparaginase.

Article Snippet: PHGDH siRNA were from Thermo Scientific Dharmacon (ON-TARGET plus Human PHGDH, smart pool) and PHGDH shRNA was from Origene.

Techniques: Expressing, Transfection, Control, shRNA

Journal: Oncotarget

Article Title: Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth.

doi: 10.18632/oncotarget.6426

Figure Lengend Snippet: Figure 6: PHGDH silencing and serine deprivation inhibit leukemia cell growth and reinforce the effects of inhibitors of Gln metabolism. Bar graphs represent the growth (%) of HL-60 leukemia cell clones expressing either control or PHGDH shRNA and incubated for 48 hours in a medium with or without (A) L-asparaginase, (B) GPNA, (C) CB-839 and (D) BPTES (n = 3). Kaplan-Meier curves depicting the impact of a serine/glycine-free (vs. control) diet on the survival of mice injected with Ba/F3 leukemia cells (E) without any other treatment (***P < 0.001, n = 9 mice per group) or (F) including a daily i.p. administration of 10 mg/kg BPTES (from day 5 to 15) (*P < 0.05, n = 6 mice per group).

Article Snippet: PHGDH siRNA were from Thermo Scientific Dharmacon (ON-TARGET plus Human PHGDH, smart pool) and PHGDH shRNA was from Origene.

Techniques: Clone Assay, Expressing, Control, shRNA, Incubation, Injection