Proliferation, cell cycle, and apoptosis of SASH3-mutated T cells after in vitro stimulation with mitogens. (A) Top: Representative plots showing Cell Trace Violet (CTV) staining in CD4⁺ or CD8⁺ T cells from P2 (red line) and control (solid gray). Red numbers correspond to the frequency of CTV^low proliferating cells (black bar). Bottom: Cumulative percentage of CTV^low cells among CD4⁺ or CD8⁺ T cells from controls (solid gray) or SASH3-mutated patients (red) in resting conditions or upon stimulation with anti-CD3 and anti-CD28, anti-CD3 and anti-CD28 plus IL-2, or PMA and ionomycin. Response to PMA and ionomycin was not studied in P3 because of a lack of available cells. Bars represent mean values ± standard error of the mean. (B) Cell cycle analysis. PBMCs from controls (CTRL1 and CRTL2) and patients (P1, P2, P4) were stimulated with anti-CD3 and anti-CD28 for 96 hours, or with PMA and ionomycin for 72 hours and then stained with 5-ethynyl-2′-deoxyuridine (EdU) and DAPI. A decreased proportion of cells in S phase and an accumulation of cells in G₂/M phase were observed in the patients. (C) Analysis of cell apoptosis. PBMCs from controls (CTRL1 to CTRL3) and patients (P1, P2, P4) were either left unstimulated or were cultured with anti-CD3 and anti-CD28 or PMA and ionomycin for 96 hours and stained with annexin V and DAPI for 30 minutes; live cells were counted by flow cytometry. P3 was not studied because of lack of available cells. Increased apoptosis was observed in all patient samples when compared with controls. Statistical analysis was performed by comparing the percentage of annexin V⁺ cells in patients vs controls. *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.