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. 2022 Mar 7;221(4):e202105107. doi: 10.1083/jcb.202105107

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© 2022 Varadarajan et al.

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Figure S3. — IP₃R-mediated calcium release is not required for calcium flashes and F-actin accumulation at TJ breaches. (A) Live imaging of calcium (GCaMP6m, green) and active Rho probe (mCherry-2xrGBD, magenta) in the animal cap epithelium of gastrula-stage Xenopus embryos treated with 20 µM BAPTA-AM after vitelline removal. Montage shows recurring calcium waves within a span of ∼500 s. The first calcium wave starts at time 0 s, and the second calcium wave starts at ∼200 s. (B) Frequency of Rho flares in vehicle (DMSO) and calcium-chelated (BAPTA-AM + 2-APB) embryos. Frequencies from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using paired two-tailed t test; n = 5 embryos, 5 experiments. (C–C″) Time-lapse images (FIRE LUT) of calcium probe (GCaMP6m) during laser wounding in embryos treated with vehicle (1% DMSO) or 10 µM XeC (IP₃R blocker) for 1 h before imaging. (C) Montage shows that blocking IP₃R (XeC) reduces the intensity and spread of calcium from the site of laser-induced wounding to the neighboring cells compared with vehicle control. White dotted circle represents the site of laser wounding. Time 0 s represents the time of laser wounding. (C′) Schematic shows the regions quantified over time in C″. (C″) Graphs show that the normalized intensity of calcium after laser wounding was reduced in XeC-treated embryos compared with vehicle controls in all three regions shown in C′. Error bars represent mean ± SEM. Vehicle: n = 4 embryos, 2 experiments; XeC: n = 6 embryos, 2 experiments. (D) Time-lapse images (FIRE LUT) of calcium probe (R-GECO1) and F-actin probe (Lifeact-GFP) during laser-induced TJ injury in embryos treated with vehicle (1% DMSO) or 10 µM XeC (IP₃R blocker) for 1 h before imaging. Local calcium increase (white dotted circles) precedes F-actin increase (white arrowheads) at the site of junctional laser injury (yellow arrowheads). Montage shows that the local calcium increase and F-actin accumulation at the site of TJ damage were comparable between vehicle- and XeC-treated embryos. Time 0 s represents time of junctional injury. (E) Quantification of experiments shown in D. Graph of mean normalized intensity shows that both intensity and time of calcium increase and F-actin accumulation following the laser injury in XeC (dotted lines) treatment were comparable to vehicle controls (solid lines). Shaded region represents SEM. Bracket represents the time interval between the peaks of calcium and F-actin. Vehicle: n = 31 flares, 14 embryos, 3 experiments; XeC: n = 27 flares, 12 embryos, 3 experiments.