Fig. S2

SCAT3 reports Caspase-3 activation. (A–C) RGC arbor expressing SCAT3 showing ECFP channel (A), Venus channel (B), and IMD ratio images (C). Caspase activation is represented by the pseudocolors that correspond to the Venus/ECFP ratio (1.5–0.5). Red represents low caspase μmactivation, and violet represents high caspase activation. (D) Quantification of arbor parameters on the Venus channel as a percentage of the IMD ratio image. SCAT3 is activated upon Caspase-3 activation. (E–H) Representative IMD ratio images of 80-hpf live tectal cell bodies. (E–H) SCAT3 (E and F) and control (G and H), before (T0; E and G) and after (F and H) the application of PAC-1 (3 h). (O and P). Quantification of Venus/ECFP ratios. 22–33 tectal cells were analyzed per condition. Dorsal views are shown, and anterior is up. Acceptor photobleaching indicates that FRET occurs between the ECFP and Venus moieties of SCAT3. (I–L) Representative IMD ratio images of 80-hpf live RGC arbors. SCAT3 (I and J), control (K and L), and tectal cell body (M and N) expressing SCAT3, before (SCAT3 T0; I, K, and M) and after (J, K, and N) 5-min photobleaching with a 514-nm laser (SCAT 3 514 nm 5 min). (O–S) Quantification of Venus/ECFP ratios across whole arbors or tectal cells. (A–C and I–L) White asterisks indicate the parent axon of RGC arbors. (E–H, M, and N) White asterisks indicate the primary dendrite of tectal cells. Five RGC arbors and 11 tectal cell bodies were analyzed per condition. Error bars represent SEMs. **, P < 0.01; ***, P < 0.001. Bars, 10 μm. The images in A–C of a SCAT3-expressing arbor are shown again in Figs. 1 E and 5 A.