Fig. 3

Protection assay of A. millepora Bcl-2 family proteins in response to extrinsic apoptotic stimuli. a Protection assay of coral Bcl-2 family proteins transfected in human HeLa cells against Bid-induced apoptosis. Plasmid constructs encoding A. millepora Bcl-2 family proteins (AmBcl-RAMBO, AmBclWA, AmMcl1-like, AmBclWD and AmBclWB), which were fused with EGFP, and an anti-apoptotic mouse Bcl-XL protein, MmBclXL, and the pCAG-mCherry plasmid were co-transfected into HeLa cells with or without pCS2-tXlBid, which encodes a potent apoptotic inducer, tXlBid. After 2 days of growth, cultures were washed to remove floating cells, and then fixed; viability was determined by monitoring mCherry-positive cells. Both phase-contrast and red fluorescence images were captured for each field under the microscope. Scale bars represent 100 μm. b A summary of the apoptosis protection assays. The numbers of mCherry-positive transfected and untransfected cells were counted at three distinct areas of each sample plate under the microscope, and the percentages of mCherry-positive cells to total cells were calculated. The number in parentheses indicates the ratio of the plasmid mixture. Data are presented as the means and standard deviations of samples counted from three independent experiments. An asterisk indicates p < 0.05 (Student’s t-test)