Fig. 1.

PCD detection in WT and CAG-sA5-YFP transgenic mESCs. a Scheme of the CAG-sA5-YFP construct to visualize PCD cells in mice. CAG chicken β-actin promoter with CMV enhancer, s secretion signal, A5 Annexin V, and YFP yellow fluorescent protein. b Transgenic mESCs stably expressing sA5-YFP (transmitted light and fluorescence pictures, left and right panels, respectively). Note weak intracellular background fluorescence of YFP overlapping with clusters of mESCs (white arrow); bright spots mark PCD cells. c Gene expression levels of sA5-YFP measured in a stable expressing mESC clone compared with wild-type G4 and mESC clones expressing YFP. d Representative fluorescent images revealing the accumulation of sA5-YFP in the membrane of PCD cells and colocalization of YFP⁺ (green) and cCasp3⁺ (magenta) signals after PCD induction by 40 µM 4-OHT (n = 3). Upper panel displays WT mESCs 24 h after PCD induction by 40 µM 4-OHT with cCasp3 signals, middle panel sA5-YFP transgenic mESCs without PCD induction, and lower panel sA5-YFP transgenic mESCs 24 h after PCD induction. e Quantification of sA5-YFP fluorescence intensities (FI) from c: FI of YFP significantly increased 24 h upon PCD induction by 4-OHT addition (n = 2). f Time-lapse microscopy (one frame every 5 min) monitoring onset and fate of PCD cells in WT mESCs preincubated prior to the experiment with 1 µM Annexin V-FITC (upper row) for 30 min and transgenic sA5-YFP mESCs (lower row) upon PCD induction (4-OHT at 40 µM for 24 h, n = 3). Examples of PCD cells labeled by Annexin V-FITC or sA5-YFP are depicted by arrows. G4 (−) G4 mESC line, YFP (+) mESC line expressing EYFP, 4-OHT 4-hydroxytamoxifen, TL transmission light, sA5-YFP secreted human Annexin V-yellow fluorescent protein, and cCasp3 cleaved caspase 3. Data in e are shown as mean ± s.e.m., ***P < 0.001 (Kruskal–Wallis statistic with Dunn’s multiple comparison post hoc test), n = 2. Bars are 50 µm (b, c) and 20 µm (f)