plasmax tm cell culture media  (CancerTools Org)

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