Fig. 3: Polydesaturation of lipids causes ER stress-related apoptosis in ESCs.

ESCs (129/black 6 background in A–I; V6.5 in J, K) were treated with the specific D6D inhibitor SC-26196 (0.2 µM), the D5D inhibitor sesamin (10 µM), DMSO control, SiRNA or its control, for 48 h in feeder free culture. Cell counting was carried out using CytoSmart cell counter and validated by manual counting (A; n = 4). Fluorescence was quantified in Oct4-GFP labeled 129/black 6 ESC culture using a plate reader (B; n = 4). Colony numbers per field were estimated under the microscope (C; 5 fields taken for each well, for 4 wells). Cell survival was further evaluated by a clonogenicity assay (D; (n = 48). Cell count was further carried out in defined Tx medium (E; n = 4). Dose response of the influence of D6D inhibition on cell proliferation rate was evaluated with 0.1–3 μM SC-26196 (F; n = 4). For validation of the specific effect of D6D, siRNA was applied (G; n = 4). The expression of IRE1, ATF6, and ASP was assessed by RT-qPCR upon D6D inhibition for 24 h (H; n = 4). The percentage of dead cells in D6D inhibited ESCs was further assessed by propidium iodide (PI) staining (I; n = 4). To evaluate apoptosis in V6.5 ESCs (with no GFP labeling) following 24 h of D6D inhibition, we used a kit based on annexin V-FITC/PI staining. A representative dot plot and the corresponding bar graph of one of four experiments (n = 4 for each) is presented J, K. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.