Fig. 4: TSP1 prevents senescence escape.

a After CIS induction, cells were transfected with siRNAs targeting TSP1. Transfection efficacy was assessed by evaluating THBS1 mRNA expression using quantitative RT-PCR or western blot analysis. Following a 24-h incubation, 10% FBS was added to induce emergence (Kolmogorov–Smirnov test, ** = p < 0.01, *** = p < 0.001). b After CIS induction, LS174T cells emergence was induced using 10% serum in the presence or absence of TSP1 (10 µg/ml, one representative image out of three experiments). c Cells were incubated for 5 mn with the indicated antibodies (B6H12 blocking antibody that prevents CD47–TSP1 binding, or its non-blocking control 2D3 or IgG1k isotype, all at 10 µg/ml). TSP1 (5 μg/ml) was then added to LS174T cells and clonogenic assays were performed for 7 days before crystal violet staining (n = 4 +/− sd, Kolmogorov–Smirnov test, * = p < 0.05). d Following treatment, emergence was induced using the secretome from PLC cells supplemented with 10% FBS and 10 µg/ml antibodies directed against CD47 or the control IgG1k isotype (n = 3 +/− sd, Kolmogorov–Smirnov test, * = p < 0.05). e MRM analysis and validation of TSP1 levels in patients that relapsed or not following chemotherapy treatment. Samples were obtained before chemotherapy treatment, see supplementary Figure 4 for the description of the clinical trial. f Analysis of platelet count in patients that relapsed or not following chemotherapy treatment