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Fig. S1. Characterization of 1-{1-(6-nitrobenzod1,3dioxol-5-yl)ethoxymethyl}-3-(pyridin-4-yl)-1H-indole (caged Rockout, cRO). Characterization of cRO was as follows. 1H NMR (300 MHz, CDCl3): δ=1.46 (d, J=6.0 Hz, 3 H), 5.22 (q, J=6.0 Hz, 1 H), 5.53 (q, J=13.8 Hz, 2 H), 5.76 (s, 1 H), 5.93 (s, 1 H), 6.86 (s, 1 H), 7.16-7.30 (m, 4 H), 7.39-7.47 (m, 3 H), 7.82 (d, J=7.5 Hz, 1 H), 8.61 (d, J=4.8 Hz, 2 H). 13C NMR (75 MHz, CDCl3): δ=23.8, 73.1, 75.8, 102.9, 104.4, 106.5, 110.5, 115.8, 119.8, 121.7, 123.6, 126.3, 126.8, 136.5, 137.4, 141.5, 142.8, 146.9, 150.3, 152.1. HRMS: m/z calculated for C23H19N3O5 M+H+: 418.1397; found: 418.1750.
Fig. S2. Hydrolysis stability test of cRO. The stability of cRO to hydrolysis under physiological conditions was tested by stirring at room temperature for 12 hours in 2 ml aq. PBS buffer:tetrahydrofuran (THF) (1:1), pH 7.4. The volatiles were evaporated and the crude mixture dissolved in deuterated DMSO (d6-DMSO) and analyzed by NMR spectroscopy. (A) 1H NMR of cRO in d6-DMSO prior to hydrolysis test. (B) 1H NMR in d6-DMSO of cRO after hydrolysis test. No decomposition of cRO was observed. TMS, tetramethylsilane (internal standard).
Fig. S3. Decaging of cRO. Decaging was confirmed by irradiating a solution of cRO in a quartz cuvette using a hand-held UV lamp (23 W, Spectroline) and analyzing the solution by HPLC/MS. Shown in each set of spectra (top to bottom): UV trace (254 nm), MS file, extracted ion MS chromatogram for 225 Da (corresponding to RO), and extracted ion chromatogram MS for 418 Da (corresponding to cRO). (A) 0.1 mM cRO in methanol (no UV). (B) 0.1 mM cRO in methanol after UV irradiation (365 nm, 15 minutes, 23 W). (C) 0.1 mM standard of non-caged RO in methanol. The data show complete disappearance of cRO after UV irradiation and 84% recovery of free RO (based on integration of peak areas, compared with 0.1 mM RO standard).
Fig. S4. cRO affects actin polymerization and protrusions in a light-dependent manner. (A-F) NIH3T3 cells were untreated (A) or exposed to DMSO (B,C), cRO (D,E) or RO (F) and left in the dark (A,B,D,F) or exposed to UV irradiation (C,E). Actin architecture is visualized with Alexa Fluor 488-phalloidin.
Fig. S5. cRO locally affects tissue-elongating gut cell migration patterns in a light-dependent manner. (A-D) Embryos expressing the green-to-red photoconvertible protein EosFP (Wacker et al., 2007) were exposed to solvent alone (A,B) or 15 µM cRO (C,D), rinsed, and irradiated on the left side of the prospective gut tube at stage 38. Embryos were then cultured in the dark until stage 43-44 and photographed in bright-field (A,C) and fluorescent (B,D) views. In embryos exposed to solvent alone the gut elongates normally and the irradiated cells are longitudinally distributed in a narrowed line along the length of the gut tube (dashed arrow, A,B), indicative of normal tissue-elongating cell rearrangements. By contrast, in the shortened cRO-treated guts, the irradiated cells have not rearranged and remain in a short, broad distribution pattern (arrows, C,D).