sHLA-G1 induces apoptosis of endothelial cells. (A) Time-lapse digital image microscopy of HUVECs after treatment with sHLA-G1. Apoptotic morphology could be detected over time, as evidenced by cytoplasmic retraction and a phase-bright appearance and membrane blebbing and blistering. These apoptotic changes are also illustrated in Videos S1 (sHLA-G1) and S2 (control). Images were visualized using an Olympus 1X70 microscope equipped with a UPlanF1 4×/0.13 NA objective. Images were captured using a Hamamatsu C4742-95-12NR camera and Image Pro Plus software version 4.5.1.29 (Media Cybernetics). (B) Kinetics curve of apoptosis induction. HUVECs were un-treated (control) or were incubated with sHLA-G1 (sG1; 1 μg/mL) or sHLA-G1mono (sG1mono; 1 μg/mL). Time-lapse microscopy was carried out to assess the appearance of apoptotic morphology. Although data were obtained every 15 minutes, data points are shown at 2-hour intervals for clarity. Mean ± SEM of pooled data from 4 experiments are shown. P < .001 between sHLA-G1 and sHLA-G1mono or control at the 50-hour time point, as determined by repeated-measures ANOVA with Tukey posttest. (C) Kinetics curve of apoptosis induction of trophoblast cells after incubation with sHLA-G1 compared with untreated (control) cells. Mean ± SEM of pooled data from 4 experiments is shown. Nonsignificance between sHLA-G1 and control at the 50-hour time point, as determined by the Mann-Whitney U test (P = .114) or paired t test (P = .127). (D) SGHEC-7 endothelial-cell apoptosis induction by sG1 (0.1 μg/mL), compared with untreated (control) cells, in the presence or absence of the caspase inhibitor zVAD-fmk assessed by time-lapse microscopy. Mean ± SEM of pooled data from 4 experiments is shown. Area under the curve was calculated from the kinetics curves. ^**P < .001, ANOVA. (E) Western blot analysis of p85 cleaved PARP expression. SGHEC-7 endothelial cells were incubated in the absence (control) or presence of sG1 (0.1 μg/mL) for 60 hours (confluent monolayer).