Extended Data Fig. 7: Cell morphological changes and activity measurement of caspase family members after photo-activation of caspase-3.
From: Time-resolved protein activation by proximal decaging in living systems
a, Apoptosis of HEK293T cells after photo–induced caspase-3 activation. Scale bars for all frames, 10 μm. n = 2. b, Apoptosis of HeLa cells after caspase-3 activation. Scale bars of all frames, 5 μm. In both cells, morphological changes upon apoptosis were observed as early as 30 min after photo-activation of caspase-3. n = 2. c, Experimental validation revealed that incorporation of ONBY at M61 blocked caspase-3 activity, which can be efficiently rescued after photo-decaging. Mean ± s.d.; n = 3; two-tailed t-test. d, Schematic of the experimental design showing that the activities of nine caspase family members were measured in HEK293T cells with either overexpression or temporal photo-activation of caspase-3. e, Normalized activity of each caspase from control cells (red), cells with transient overexpression of wild-type (WT) caspase-3 (blue), cells with overexpression of caspase-3-M61-ONBY before (green) and after (orange) photo-activation. Overexpression of the wild-type caspase-3 resulted in markedly increased activities for most of the other caspases, whereas direct photo-activation of the caspase-3 variant enabled by CAGE-prox only activated this specific enzyme without much influence on other caspases. It allows the temporal profiling of proteolytic substrates immediately after caspase-3 activation. Error bars represent mean ± s.d.; n = 3; two-tailed t-test. f, Replotting of d with activities of all caspase members shown in the same scale. Error bars represent mean ± s.d.; n = 3. All above-mentioned samples are biological replicates. P values are shown in the figure.
