Fig 1: APG-115 increase mouse T cell proliferation and enhances mouse CD4+ T cell activation. a CD4+ T and CD8+ T cells were positively selected from mouse spleens using magnetic beads and then stimulated with indicated concentrations of plate-bound anti-CD3 and 2 μg/mL anti-CD28 in the presence of 250 nM APG-115 or DMSO. After 72 h, relative cell numbers were determined using CellTiter-Glo luminescent cell viability assay (Promega) and normalized to unstimulated cultures treated with DMSO control. * P < 0.05. b immunoblots for the expression of caspase 3, cleaved caspase 3, and Zap-70 (loading control) in total cell lysates of anti-CD3/CD28-stimulated CD4+ T cells exposed to APG-115 or solvent control DMSO for 3, 6, or 24 h (h). c CD4+ T cells were positively selected from mouse spleens using magnetic beads and then stimulated with 10 μg/mL plate-bound anti-CD3 and 2 μg/mL anti-CD28 in the presence of 250 nM APG-115 or DMSO for the indicated periods of time. T cell activation markers (CD25 and CD62L) were determined by flow cytometry. CD25high CD62Llow T cells represented an activated population. d an increase in cell size was shown after APG-115 treatment
Fig 2: In vitro efficacy analysis of the bispecific ITs using CellTiter‐Glo® Luminescent Cell Viability Assay (Promega, cat# G7571) to human CD25+CCR4+ T‐cell lymphoma cell line Hut102/6TG. (a) C21 IT as negative control (black line); (b) IL2 fusion toxin alone (blue line); (c) CCR4 IT alone (red line); (d) IL2‐CCR4 bispecific IT (orange line); and (e) CCR4‐IL2 bispecific IT (green line). Y‐axis: inhibition rate of the cell viability by determining the number of viable cells based on the quantification of the ATP present. X‐axis: plated IT concentration. Cycloheximide (1.25 mg·mL−1) was used as a positive control. The negative control contained cells without IT. Data were from three independent assays. Statistical analysis was performed using two‐way ANOVA (n = 3). Error bars indicate ± SD.
Fig 3: Early but not late ferroptotic cancer cells induce immunogenic cell death in vitro. (A) Cell-death recovery was measured by Sytox Green fluorescence in cancer MCA205 cells on stimulation with RSL3. After 1 hour, 3 hours and 6 hours of RSL3 stimulation, the cells were washed in PBS and reseeded in fresh culture medium (without RSL3) and cultured further for 23 hours, 21 hours or 18 hours, respectively, to give a total culture time. (B) Schematic representation of the measurement of ATP and HMGB1 release in the coculture of BMDCs with ferroptotic MCA205 cells shown in figure 6C, D. Viable or dying cancer MCA205 cells (induced with either RSL3 or MTX as positive controls) were harvested and cocultured with BMDCs for 3 hours (for ATP) or 24 hours (for HMGB1). Next, the supernatants were collected and analyzed for ATP using CellTiter-Glo Luminescent Cell Viability Assay (Promega) and for HMGB1 with ELISA kit (IBL-Hamburg). (C) The concentration of ATP in the coculture of BMDCs with ferroptotic MCA205 cells after 3 hours of coincubation. The values are the means±SEM of four independent measurements performed in triplicates. Two-way ANOVA was used to calculate the statistical significance (*p<0.05, **p<0.01). (D) The concentration of HMGB1 in the coculture of BMDCs with ferroptotic MCA205 cells after 24 hours. The values are the means±SEM of three independent measurements performed in duplicates. One-way ANOVA with Tukey’s multiple comparison test was used to calculate the statistical significance: ****p<0.0001. For positive control, BMDCs were cocultured with MCA205 cells killed by MTX (2 µM, 24 hours). Activation and maturation profiles of BMDCs from these experiments are shown in figure 3A–C. BMDCs, bone-marrow derived dendritic cells; HMGB1, high-mobility group box 1; MTX, mitoxantrone; PBS, phosphate-buffered saline; RSL3, RAS-selectivelethal 3.
Fig 4: Fibroblasts from sPD patients treated with AntiOxCIN4presented increased metabolic activity and mitochondrial maximal respiration. Cellular proliferation (A) and metabolic activity (B) were measured using the sulforhodamine B (SRB) and resazurin assays, respectively. Intracellular ATP levels (C) were measured by using CellTiter-Glo Luminescent Cell Viability Assay (Promega) following manufacturer's instructions. The Seahorse XFe96 Extracellular Flux Analyzer was used to measure Oxygen Consumption Rate (OCR) (D) and extracellular acidification rate (ECAR) (E). Several OCR parameters were evaluated: basal cell respiration (F), maximal cell respiration (G) and ATP production-linked OCR (H) and different metabolic parameters (J). Energy map showing the metabolic potential of cells before adding of oligomycin (time point 3) and after being stressed with oligomycin plus FCCP (time point 7) (I). Each measurement corresponds to one different individual (5 fibroblasts from sPD patients and 5 fibroblasts from respective sex- and age-matched healthy controls). Data was normalized by the control condition (C = 100%). Data are expressed as mean ± SEM of 5 independent experiments. Statistical significance was accepted with (*) p < 0.05, (**) p < 0.01, (***) p < 0.005 to C vs PD or C + AntiOxCIN4 vs PD + AntiOxCIN4 and (#) p < 0.05, (###) p < 0.005 to C vs C + AntiOxCIN4 or PD vs PD + AntiOxCIN4.
Fig 5: In vitro efficacy analysis of the CD19 immunotoxins using CellTiter‐Glo® Luminescent Cell Viability Assay (Promega, cat# G7571) to human CD19+ mantle cell lymphoma cell line JeKo‐1. (a) Monovalent anti‐human CD19 immunotoxin [DT390‐scFv (FMC63), green line]; (b) bivalent anti‐human CD19 immunotoxin [DT390‐BiscFv (FMC63), red line]; (c) single‐chain foldback diabody anti‐human CD19 immunotoxin (blue line); (d) DT390 alone (purple line). Y‐axis: inhibition rate of the cell viability by determining the number of viable cells based on the quantification of the ATP present. X‐axis: plated anti‐human CD19 immunotoxin concentration. Cycloheximide (1.25 mg·mL−1) was used as a positive control. The negative control contained cells without immunotoxin. p < 0.0001 by log‐rank (Mantel–Cox) test of Prism (n = 3). Error bars indicate ±SD. Data are representative of multiple assays.
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