Fig. 8

Ca²⁺ improve the capacity of ENO to cause apoptosis. A Representative images of droplet formation of GFP-RPSA in vitro (Scale bar = 10 μm). One group additionally had added 3 mM Ca²⁺. The area of RPSA condensates were used for quantitative analysis by Image J (RPSA group: n = 72 condensates, RPSA + Ca²⁺ group: n = 83 condensates, mean ± median). Mann–Whitney test. B After HCMCE/D3 cells were pretreated with or without Ca²⁺ (200 μM) medium for 24 h, RPSA on the cell surface was observed by immunofluorescence staining using the antibody against RPSA. Representative confocal images are shown (scale bar = 40 μm) and magnified (scale bar = 20 μm). C HEK-293 T cells overexpressing EGFP-RPSA^WT for 12 h. The transfected cells were pretreated as described in (B). FRAP analyses of condensates in each group. Representative FRAP images are shown (scale bar = 20 μm). In Fig. 1, the symbols for "CI" and "Bleached" were depicted. Quantification of FRAP as mean ± SD (n = 10 condensates). D After HCMCE/D3 cells were pretreated as described in (B), ENO (30 μg/mL) protein was added to the cells. After 24 h, cells were treated with or without 1,6-Hex. The amount of ENO protein acting on the cell was quantified by western blot analysis. Quantification was performed by Image J as mean ± SD (n = 3 biologically independent samples). E and F The procedure followed by Ca²⁺ chelating agent experiments (E a) and mitochondrial protective agent experiments (F a) is shown. (E b, F b) Flow cytometry analyzed that the death level of cells. The ratio of dead cells to total cells in the indicated conditions was analyzed by FlowJo as mean ± SD (n = 4 biologically independent samples). A, D-F NS for not significant, * for P < 0.05, *** for P < 0.001; D-F one-way ANOVA with Tukey’s test