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. 2019 Dec 30;21(1):267. doi: 10.3390/ijms21010267

Figure 3.

Figure 3

Mouse PHLDA1 can induce caspase-7 (but not caspase-3) activation and detachment of cells. (a) Mouse NIH3T3 cells were transiently transfected with vectors coding for indicated fusion proteins and protein extracts were analyzed by western blot. NT—not transfected cells, TR—transfection reagent only. ACTB and HSPA8 were used as loading controls. Active caspase-3 and caspase-7 were quantitated based on densitometry of replicated blots and results are shown in corresponding charts; asterisks indicate statistically significant (* p < 0.05) differences against the EGFP-transfected cells. (b) An example of the live-cell imaging of NIH3T3 cells transiently transfected with the PMAIP1/EGFP construct showing the apoptotic membrane blebbing in a cell overexpressing PMAIP1, while control cells (transfected with the EGFP vector only, right photo) were not affected. (c) An example of the live-cell imaging of NIH3T3 cells transiently transfected with vectors coding for PHLDA1/EGFP, EGFP/PHLDA1 or EGFP showing detachment of cells overexpressing PHLDA1. Images were recorded in EGFP (green) and brightfield channels for two days. The observation period shown for each vector is given above the panels. The relative time of acquisition is displayed in the upper right corner of each image.