plasmax  (CancerTools Org)

Journal: bioRxiv

Article Title: hENT Inhibition Prevents Pyrimidine-Driven Resistance to DHODH Inhibition in Malignant Rhabdoid Tumors

doi: 10.64898/2026.01.25.701565

Figure Lengend Snippet: A) Overview of the concentrations of nucleosides and nucleobases in Advanced DMEM/F12 (base medium for KOM) and Plasmax™ according to the manufacturer. “NA” indicates that the respective compound is absent from the medium formulation. Shades of red represent relative concentrations of each component. B) Dose-response curves for ecMRT tumoroid model 60T treated with BAY or GTX-196 for 120 hours in standard KOM, KOM supplemented with 3 μM uridine, or Plasmax. Data represent mean ± SD from three independent experiments. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). C) Relative abundance of different isotopologues in ADP, ATP, GDP, and GTP in two different ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose in the presence of 0⍰μM, 3⍰μM, or 30⍰μM uridine. All conditions were statistically compared to the 0⍰μM uridine condition. D) Total abundance and isotopic labeling pattern of CDP and UDP in two ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine. Statistical testing was performed within each uridine condition. E) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with 5 nM GTX-196 or DMSO vehicle. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl within each uridine condition. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: Single cells were plated at a density of 2.000 cells/μL in 5 μL 75% BME droplets topped with KOM PL and Plasmax (CancerTools, 156371) (see exact formulations in ) in a pre-warmed flat-bottom 96-well plate (Greiner, 655-160).

Techniques: Formulation, Incubation, Isotopic Labeling

Journal: bioRxiv

Article Title: hENT Inhibition Prevents Pyrimidine-Driven Resistance to DHODH Inhibition in Malignant Rhabdoid Tumors

doi: 10.64898/2026.01.25.701565

Figure Lengend Snippet: A-B) Bar graphs depicting the average cell viability (%) of ecMRT tumoroid models following 120-hour treatment with 1 μM BAY-2402234 (BAY) (panel A ) or 1 μM GTX-196 (panel B ) in the presence of standard KOM or Plasmax™ medium. Data represent the mean ± standard deviation (SD) of n = 3 independent experiments, each performed in technical duplicates. Viability values were normalized to the DMSO vehicle control in KOM (set to 100%). C) Schematic overview illustrating the incorporation of carbon atoms from uniformly labeled [U- 13 C 6 ]-glucose (red dots) and [U- 13 C 9 ]-uridine (yellow dots) into pyrimidine nucleotide UTP via the de novo nucleotide biosynthesis and nucleotide salvage pathways. In the case of [U- 13 C 6 ]-glucose, the ribose moiety (in orange) is synthesized via the pentose phosphate pathway (PPP), resulting in UTP labeled at five carbons ([ 13 C 5 ]). Additional carbons are contributed by aspartate (ASP) to the pyrimidine ring (in purple), generating UTP isotopologues with up to [ 13 C 8 ] labeling. In contrast, [ 13 C 9 - 15 N 2 ]-uridine is taken up through the salvage pathway as an intact molecule, yielding fully labeled UTP ([ 13 C 9 - 15 N 2 ]). Partial catabolism of [ 13 C 9 - 15 N 2 ]-uridine can lead to differential labeling patterns: UTP [ 13 C 5 - 15 N 0 ] indicates salvage of the labeled ribose with replacement of the pyrimidine nucleobase via de novo synthesis, while UMP [ 13 C 4 - 15 N 2 ] reflects incorporation of a labeled uracil base with an unlabeled ribose, suggesting base salvage following uridine breakdown. D-E) Dose-response curves for ecMRT tumoroid models treated with BAY (panel D ) or GTX-196 (panel E ) for 120 hours in standard KOM, or KOM supplemented with 3 μM uridine or 30 μM uridine. Data represent mean ± SD from three independent experiments, each consisting of technical quadruplicates. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). F) Total abundance and isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose in the presence of 0⍰μM, 3⍰μM, or 30⍰μM uridine upon 48-hour treatment with DMSO vehicle or 5 nM GTX-196. Unless specified otherwise, statistical comparisons were performed within each uridine condition. G) Fraction of unlabeled ([ 13 C 0 ]) CDP, CTP, UDP, and UTP in two different ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose in the presence of 0⍰μM, 3⍰μM, or 30⍰μM uridine. All conditions were statistically compared to the 0⍰μM uridine condition. H) Total abundance and isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with DMSO vehicle or 5 nM GTX-196. Unless specified otherwise, statistical comparisons were performed within each uridine condition. I) Fraction of [ 13 C 9 - 15 N 2 ]-labelled CDP, CTP, UDP, and UTP in two different ecMRT tumoroid models following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: Single cells were plated at a density of 2.000 cells/μL in 5 μL 75% BME droplets topped with KOM PL and Plasmax (CancerTools, 156371) (see exact formulations in ) in a pre-warmed flat-bottom 96-well plate (Greiner, 655-160).

Techniques: Standard Deviation, Control, Labeling, Synthesized, Isotopic Labeling, Incubation

Journal: bioRxiv

Article Title: hENT Inhibition Prevents Pyrimidine-Driven Resistance to DHODH Inhibition in Malignant Rhabdoid Tumors

doi: 10.64898/2026.01.25.701565

Figure Lengend Snippet: A) Box-plots displaying the log 10 -transformed 13 C-yeast extract-normalized peak areas of the metabolites uridine (left), uracil (middle), and hypoxanthine (right). See for proxy-metabolites used for normalization. Each dot represents the normalized peak area from an individual sample. Sample colors correspond to sample type and tumor metastatic status. The gray dashed line indicates the log 10 -normalized peak area measured in Plasmax™ reference samples containing known metabolite concentrations.

Article Snippet: Single cells were plated at a density of 2.000 cells/μL in 5 μL 75% BME droplets topped with KOM PL and Plasmax (CancerTools, 156371) (see exact formulations in ) in a pre-warmed flat-bottom 96-well plate (Greiner, 655-160).

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: hENT Inhibition Prevents Pyrimidine-Driven Resistance to DHODH Inhibition in Malignant Rhabdoid Tumors

doi: 10.64898/2026.01.25.701565

Figure Lengend Snippet: A) Schematic overview of de novo nucleotide biosynthesis and nucleotide salvage pathways. Nucleosides and nucleobases are imported into the cell via hENT 1 and 2 and subsequently phosphorylated to their corresponding nucleotide monophosphates (NMPs). In parallel, nucleotides can be synthesized de novo from metabolic precursors, resulting in the formation of NMPs. Key enzymes involved in these pathways, including uridine-cytidine kinase (UCK), DHODH, and carbamoyl-phosphate synthetase 2/ aspartate transcarbamylase/ dihydroorotase (CAD), are highlighted. Both pathways can be subjected to pharmacological inhibition by agents targeting hENT1/2 (e.g., dipyridamole) and DHODH (e.g., BAY-2402234 and GTX-196). B) Dose-response curves for ecMRT tumoroid models 60T and 103T treated with BAY-2402234 for 120 hours in Plasmax™ medium, with or without dipyridamole (500 nM). ecMRT tumoroids treated with BAY-2402234 in KOM medium served as a reference. Data represent mean ± SD from three independent experiments, each performed with technical duplicates. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). C-D) Dose-response curves for ecMRT tumoroid model 103T treated with BAY-2402234 (panel C ) or GTX-196 (panel D) for 120 hours in KOM supplemented with 30 μM uridine, with or without different concentrations of dipyridamole. Data represent mean ± SD from three independent drug matrix experiments. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). E-F) ZIP synergy landscapes for BAY-2402234 + dipyridamole (E) and GTX-196 + dipyridamole (F) , corresponding to panels C–D. ZIP scores >10 indicate synergistic interactions, scores between -10 and 10 represent additive effects, and scores <10 indicate antagonism. Regions outlined in white represent the most synergistic concentration combinations. Synergy landscapes were generated using SynergyFinder software . G) Bar graph depicting the average cell viability (%) of ecMRT tumoroid models following 120-hour treatment with 5 nM GTX-196, 10 μM dipyridamole, or their combination, in the presence or absence of nucleoside supplementation. Data represent the mean ± standard deviation (SD) of n = 3 independent experiments, each performed in technical triplicates. Viability values were normalized to the DMSO vehicle control without nucleoside supplementation (set to 100%). H) Relative isotopologue distribution (fractional labeling) of CTP and UTP in two ecMRT models following 24-hour incubation with [U- 13 C 6 ]-glucose upon 48-hour treatment with DMSO vehicle or 10 μM DP, in the presence or absence of 30⍰μM uridine. Statistical testing was performed within each uridine condition. I) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with [U- 13 C 6 ]-glucose upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof, in the presence or absence of 30⍰μM uridine. DMSO and GTX-196 reference data originate from the same [U- 13 C 6 ]-glucose tracing experiment as previously presented in , but are shown again to provide direct comparison across all treatment conditions. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl within each uridine condition. J) Isotopic labeling pattern of CTP and UTP in two ecMRT tumoroid models following 24-hour incubation with 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof. DMSO and GTX-196 reference data originate from the same [ 13 C 9 - 15 N 2 ]-uridine tracing experiment as previously presented in , but are shown again to provide direct comparison across all treatment conditions. Unless specified otherwise, all conditions were statistically compared to the DMSO ctrl. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: Single cells were plated at a density of 2.000 cells/μL in 5 μL 75% BME droplets topped with KOM PL and Plasmax (CancerTools, 156371) (see exact formulations in ) in a pre-warmed flat-bottom 96-well plate (Greiner, 655-160).

Techniques: Synthesized, Inhibition, Concentration Assay, Generated, Software, Standard Deviation, Control, Labeling, Incubation, Isotopic Labeling, Comparison

Journal: bioRxiv

Article Title: hENT Inhibition Prevents Pyrimidine-Driven Resistance to DHODH Inhibition in Malignant Rhabdoid Tumors

doi: 10.64898/2026.01.25.701565

Figure Lengend Snippet: A) Bar graph depicting the average viability (%) of ecMRT tumoroids cultured in KOM supplemented with 30 μM uridine following 120-hour treatment with various concentrations of dipyridamole. Data represent the mean ± SD of n = 3 independent experiments, each consisting of technical triplicates. Viability values were normalized to the DMSO vehicle control (100%). B) Dose-response curves for ecMRT tumoroid models 60T and 78T treated with GTX-196 for 120 hours in Plasmax™ medium, with or without the hENT1/2 inhibitor dipyridamole (500 nM). ecMRT tumoroids treated with GTX-196 in KOM medium served as a reference. Data represent mean ± SD from three independent experiments, each performed with technical duplicates. Data are normalized to DMSO vehicle in KOM (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). C-D) Dose-response curves for ecMRT tumoroid models 60T treated with BAY-2402234 (panel C ) and 103T treated with GTX-196 (panel D) for 120 hours in KOM supplemented with 3 μM uridine, with or without different concentrations of hENT1/2 inhibitor dipyridamole. Data represent mean ± SD from three independent drug matrix experiments. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). E-F) Two-dimensional (2D) synergy landscapes visualized as contour plots, displaying ZIP (Zero Interaction Potency) synergy scores across concentration matrices for BAY-2402234 combined with dipyridamole (panel E ) and GTX-196 combined with dipyridamole (panel F ) (corresponding to figure panels C and D ). ZIP scores >10 indicate synergistic interactions, scores between -10 and 10 represent additive effects, and scores <–10 indicate antagonism. Regions outlined in white represent the most synergistic concentration combinations. Synergy landscapes were generated using SynergyFinder software . G , I) Dose-response curves for ecMRT tumoroid model 103T treated with GTX-196 for 120 hours in KOM supplemented with 30 μM uridine, with or without different concentrations of hENT inhibitors draflazine (panel G ) or nitrobenzylthioinosine (NBMPR; panel I). Data represent mean ± SD from three independent drug matrix experiments. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC 50 ). H , J ) Two-dimensional (2D) synergy landscapes visualized as contour plots, displaying ZIP (Zero Interaction Potency) synergy scores across concentration matrices for GTX-196 combined with draflazine (panel H ) and GTX-196 combined with NBMPR (panel J ) (corresponding to figure panels G and I ). ZIP scores >10 indicate synergistic interactions, scores between -10 and 10 represent additive effects, and scores <–10 indicate antagonism. Regions outlined in white represent the most synergistic concentration combinations. Synergy landscapes were generated using SynergyFinder software . K) Isotopic labeling pattern of extracellular uridine levels measured in the medium of BME (no cells) and two different ecMRT tumoroids following 24-hour incubation with 3⍰μM or 30⍰μM [ 13 C 9 - 15 N 2 ]-uridine upon 48-hour treatment with DMSO vehicle, 5 nM GTX-196, 10 μM DP, or the combination thereof. All conditions were statistically compared to the BME (no cells) reference group. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: Single cells were plated at a density of 2.000 cells/μL in 5 μL 75% BME droplets topped with KOM PL and Plasmax (CancerTools, 156371) (see exact formulations in ) in a pre-warmed flat-bottom 96-well plate (Greiner, 655-160).

Techniques: Cell Culture, Control, Concentration Assay, Generated, Software, Isotopic Labeling, Incubation

Journal: bioRxiv

Article Title: Thiol Scarcity in Cerebrospinal Fluid Renders Leptomeningeal Acute Lymphoblastic Leukaemia Therapeutically Vulnerable to Ferroptosis

doi: 10.64898/2025.12.15.693383

Figure Lengend Snippet: ( A ) Ratio of oxidised (510 nm) to reduced (591 nm) BODIPY 581/591 C11 in 697 cells incubated for 24 hours in Plasmax TM or Patient CSF, with or without 20 µM α-tocopherol as indicated. N = 3 independent experiments. P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (B) CHAC1 mRNA expression from publicly available RNA sequencing of ALL cells (REH) incubated in RPMI + 10% FBS or human CSF for 48 hours. P-values calculated using unpaired two-tailed Student’s t-test. (GSE274857). Error bars represent mean ± SEM. (C) Ratio of oxidised (510 nm) to reduced (591 nm) BODIPY 581/591 C11 in 697 cells incubated for 24 hours in Plasmax TM or CSFmax, with or without 20 µM α-tocopherol. N = 5 independent experiments. P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (D) qPCR quantification of CHAC1 mRNA expression, normalised to ACTB mRNA, in 697 cells treated for 48 hours in CSFmax or Plasmax TM . N = 3 independent experiments. P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (E) Schematic illustrating the mechanism of action of ferroptosis inducers RSL3 and Erastin (orange), and the ferroptosis inhibitor Ferrostatin-1 (pink). Created with BioRender.com. (F) Left Panel: Representative dose-response curves of 697 cells treated with RSL3 (GPX4i) in Plasmax TM , CSFmax, with or without 2 µM Ferrostatin-1 as indicated. Luminescence values for each concentration were normalised to vehicle-treated controls. N = 3 independent experiments. Right Panel: LogEC 50 values in Plasmax vs CSFmax. Each point indicates the calculated logEC 50 from independent experiments (n=3). P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (G) Left Panel: Dose-response curves of 697 cells treated with Erastin(XCTi) in Plasmax TM , CSFmax, with or without 2 µM Ferrostatin-1. Luminescence values for each concentration were normalised to vehicle-treated controls. N = 4 independent experiments. Right Panel: LogEC 50 values in Plasmax vs CSFMax. Each point indicates the calculated logEC 50 from independent experiments (n=4). P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (H) Upper Panel (i) : Dose-response curves of 697 cells treated with RSL3 in CSFmax with or without 30 nM Na 2 SeO 3 , with or without 65 µM L-Cystine as indicated. Luminescence values for each concentration were normalised to vehicle-treated controls. N = 3 independent experiments. Lower Panel (ii) : LogEC 50 values. Each point indicates the calculated logEC 50 from independent experiments (n=3).P-values determined by a one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent mean ± SEM. (I) Relative P.I. fluorescence of 697 cells treated with 75nM of RSL3 for 24 hours in CSFmax with or without 30 nM Na 2 SeO 3 , with or without 65 µM L-Cystine. Fluorescence normalised to lethal controls defined in the methods. N = 3 independent experiments. P-values determined by a one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean ± SEM. (J) Ratio of oxidised (510 nm) to reduced (591 nm) BODIPY 581/591 C11 in 697 cells incubated for 48 hours in CSFmax, with or without 30 nM Na 2 SeO 3 or 65 µM L-Cystine. N = 4 independent experiments. P-values determined by a one-way ANOVA with Dunnett’s multiple comparisons test relative to CSFmax alone. Error bars represent mean ± SEM. * ≤ 0.05 **** ≤ 0.0001

Article Snippet: These solutions were further diluted in a buaered solution containing sodium bicarbonate, phenol red, Plasmax TM cell culture media ( CancerTools.org , #156371) and BME vitamin mix (Sigma, #B6891).

Techniques: Incubation, Two Tailed Test, Expressing, RNA Sequencing, Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Thiol Scarcity in Cerebrospinal Fluid Renders Leptomeningeal Acute Lymphoblastic Leukaemia Therapeutically Vulnerable to Ferroptosis

doi: 10.64898/2025.12.15.693383

Figure Lengend Snippet: ( A ) Immunoblot images of LRP8 and Vinculin in 018Z transduced with NTC or with two LRP8 CRISPR guides (sg1 and sg2). (B) Left Panel: Dose-response curves of 018Z NTC or sgLRP8 #1 cells treated with Erastin in RPMI with 10%FBS. Luminescence values normalised to vehicle-treated controls. N = 3 independent experiments. Right Panel: logEC 50 values for Erastin. Each point indicates the calculated logEC 50 from independent experiments. P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (C) Cell Number of NTC or sgLRP8 #1 grown in media with or without 100 µM L-cystine over 96 hours. P-values determined by a one-way ANOVA with Tukey’s multiple comparisons test. N = 3 independent experiments. Error bars represent mean ± SEM. (D) Ratio of oxidised (510 nm) to reduced (591 nm) BODIPY 581/591 C11 in NTC or sgLRP8 #1 018Z cells incubated for 24 hours with or without 2.5 µM Erastin. N = 4 independent experiments. P-values determined by one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent mean ± SEM. (E) Relative P.I fluorescence of 018Z NTC or sgLRP8 #1 cells treated with 2.5µM Erastin for 48 hours. Fluorescence values were normalised to lethal controls described in the Methods. N = 3 independent experiments. P-values determined by a one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent mean ± SEM. (F) Immunoblot images of GPX4, LRP8 and β-Actin protein levels in sgLRP8 #1 or NTC cells cultured in media with or without 100 µM L-cystine for 48 hours. 1 experiment representative of 3 are shown. (G) Immunoblot image of SEPHS2 and β-Actin in 018Z cells transduced with NTC or SEPHS2 CRISPR guide. (H) Left Panel: Dose-response curves of 018Z NTC and sgSEPHS2 cells treated with Erastin in RPMI with 10% FBS. Luminescence values normalised to vehicle-treated controls. N = 3 independent experiments. Right Panel: logEC 50 values for Erastin. Each point indicates the calculated logEC 50 from independent experiments. P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (I) Cell number of NTC or sgSEPHS2 in media with or without 100 µM L-cystine over 96 hours. P-values determined by a one-way ANOVA with Tukey’s multiple comparisons test. N = 3 independent experiments. Error bars represent mean ± SEM. (J) Ratio of oxidised (510 nm) to reduced (591 nm) BODIPY 581/591 C11 in NTC or sgSEPHS2 018Z cells incubated for 24 hours with or without 2.5 µM Erastin. N = 3 independent experiments. P-values determined by two-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent mean ± SEM. (K) Cell death in Plasmax TM and human CSF. Flow cytometry quantification of DAPI-positive staining in NTC or sgLRP8 #1 cells cultured in Plasmax TM or human CSF for 24 hours. P-values determined by two-way ANOVA with Tukey’s multiple comparisons test. N = 3 independent experiments. Error bars represent mean ± SEM. (L) Relative levels of GPX4 intracellular staining in sgLRP8 #1 or NTC 018Z cells after 48 hours in Plasmax TM or human CSF. Data were normalised against Plasmax TM treated NTC cells. N=3 independent experiments. P-value refers to a one-sample Wilcoxon signed-rank test. (M) Schematic representation of the proposed model whereby CSF induces compensatory upregulation of LRP8 to maintain GPX4 activity ( Left panel ) and that LRP8 targeting induces a synthetic lethality in CSF ( Right panel ). Error bars represent mean ± SEM. * ≤ 0.05 ** ≤ 0.01 **** ≤ 0.0001

Article Snippet: These solutions were further diluted in a buaered solution containing sodium bicarbonate, phenol red, Plasmax TM cell culture media ( CancerTools.org , #156371) and BME vitamin mix (Sigma, #B6891).

Techniques: Western Blot, Transduction, CRISPR, Two Tailed Test, Incubation, Fluorescence, Cell Culture, Flow Cytometry, Staining, Activity Assay

Journal: bioRxiv

Article Title: Thiol Scarcity in Cerebrospinal Fluid Renders Leptomeningeal Acute Lymphoblastic Leukaemia Therapeutically Vulnerable to Ferroptosis

doi: 10.64898/2025.12.15.693383

Figure Lengend Snippet: ( A ) Auranofin chemical structure. Pubchem ID:16667669 (B) Immunoblot images of GPX4, GPX1 and Vinculin in 018Z cells treated with 150nM Na 2 SeO 3 and with an increasing concentration of Auranofin for 72 hours as indicated. The results from 1 experiment representative of 3 are shown. (C) Upper Panel: Dose-response curves of 018Z, treated with Auranofin in CSFmax or Plasmax TM as indicated. Luminescence values were normalised to vehicle-treated controls. Points indicate the mean of N = 3 independent experiments. Lower Panel: logEC 50 values for Auranofin. Each point indicates the calculated logEC 50 from independent experiments (n=3). P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (D) Relative P.I fluorescence of 018Z cells treated with 100nM Auranofin for 48 hours in Plasmax TM or CSFmax. Fluorescence values were normalised to lethal controls described in Methods. P-values determined by a one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent mean ± SEM. (E) Upper Panel: Dose-response curves of 018Z treated with Auranofin in CSFmax with or without 65µM cystine. Luminescence values normalised to vehicle-treated controls. Points indicate the mean of N = 3 independent experiments. Lower Panel: logEC 50 values for Auranofin. Each point indicates the calculated logEC 50 from independent experiments (n=3). P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (F) Leukaemic burden assessed in the spleen by measuring its weight, or in the CNS and in the BM by counting the total number of blasts. NSG mice xenografted with human 018z ALL cell lines were treated with Auranofin (Mon-Fri). N= 6 or 7 mice per cohort as indicated by points. P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. * ≤ 0.05 (G) Schematic of the patient-derived xenotransplantation and Auranofin treatment. Created with BioRender.com. (H) Leukaemic burden assessed in the spleen by measuring its weight, or in the CNS and in the BM by counting the total number of blasts. NSG mice xenografted with human 859I ALL PDX cells treated with Auranofin (Mon-Fri). P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. (I) Ratio of oxidised (510 nm) to reduced (591 nm) BODIPY 581/591 C11 in Auranofin or vehicle treated cells obtained from BM or CNS of PDX xenograft at the time of sacrifice. N=4 mice. P-values determined by a two-way ANOVA with Tukey’s multiple comparison test. Error bars represent mean ± SEM. (J) Left Panel: Immunoblot images of GPX4, full length SELENOP, truncated SELENOP and β-Actin in samples from spleen of mice treated with vehicle or Auranofin as described in N=4 mice. Right Panel: Quantification of GPX4 protein levels. P-values calculated using unpaired two-tailed Student’s t-test. Error bars represent mean ± SEM. * ≤ 0.05.

Article Snippet: These solutions were further diluted in a buaered solution containing sodium bicarbonate, phenol red, Plasmax TM cell culture media ( CancerTools.org , #156371) and BME vitamin mix (Sigma, #B6891).

Techniques: Western Blot, Concentration Assay, Two Tailed Test, Fluorescence, Derivative Assay, Comparison