Fig. 2
The N-terminal domain of Gsdma3 harbors the cell death-inducing ability of Gsdma3. (a) Immunostaining of HaCaT cells transfected with the indicated plasmid constructs. Cells were live-cell stained with Annexin V-Cy3 and 7-AAD and immunostained for the tagged constructs. Arrows, Annexin V-Cy3 cell surface staining (red); arrowheads, 7-AAD nuclear staining (red); Tag-specific staining (green). (b) Quantification of the apoptosis/necrosis staining. The percentage of Annexin V+ (early apoptosis) cells and the percentage of Annexin V+7AAD+ (late apoptosis) cells among the tag+ cells (mean + s.d., n > 5 microscopy fields from three independent experiments) are shown for each transfected construct. Vector-transfected cells exhibited 0.1 ± 0.2 % early apoptosis and 1.4 ± 0.9 % late apoptosis. (c) TUNEL staining in HaCaT cells transfected with the indicated plasmid constructs, followed by immunostaining for the tagged constructs. TUNEL (green) and tag-specific (red) staining results are shown. DAPI counterstains the nuclei in blue. Scale bar, 50 μM. (d) Quantification of the TUNEL stainings by the percentage of TUNEL+ cells among tag+ cells (mean + s.d., n > 6 microscopy fields from two independent experiments). Vector-transfected cells displayed a 1.32 ± 0.57 % TUNEL+ signal. (e, f) Flow cytometry analysis of cell death and mitochondrial membrane potential (MMP) depolarization. HEK293T cells were transfected with flag tagged-constructs and the vector pIRES-hrGFP-1a, stained with 7-AAD and DiIC1(5), followed by flow cytometry analysis. Quantifications results show the fold increase (mean + s.d., n >3) in the fraction of dead (7AAD+) GFP+ cells and mitochondrial membrane potential depolarization (DiIC1(5)low) GFP+ cells over that of pIRES-GFP-1a vector-transfected cells (Dead, 1.88 ± 0.7 %, Low MMP, 4.76 ± 2.14 %).*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant