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. 2022 Nov 18;33(14):ar136. doi: 10.1091/mbc.E22-06-0205

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© 2022 Chumki et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 3: — p115RhoGEF is required for junctional RhoA activation to maintain the apical actomyosin array, TJs, and AJs. (A-B) Live imaging of cells expressing active RhoA probe (mCherry-2xrGBD, Fire LUT) in control, p115.S+L KD, p115.S KD, and p115.S KD + p115.S wobble embryos. The data for each condition was acquired with the same settings, and the LUTs were adjusted in the same way for each. GeneTools-Blue MO(s), when present, are shown in blue (Cyan Hot LUT). (B) Ratio of junction/cytoplasm signal of RhoA is reduced with p115RhoGEF KD. Injection of p115.S wobble partially rescues junctional active RhoA intensity. p = <0.0001(**) and p = 0.003(*). Error bars represent mean ± SEM; significance was calculated using unpaired t tests. Control: n = 10 embryos, 3 experiments; p115.S+L KD: n = 10 embryos, 3 experiments; p115.S KD: n = 10 embryos, 3 experiments; p115.S KD + p115.S wobble: n = 10 embryos, 3 experiments. (C–E) (C) Sum projection of fixed staining for F-actin (Alexa Fluor 488 or 647 phalloidin), P-MLC (anti-P-MLC) in control, p115.S+L KD, p115.S KD, and p115.S KD + p115.S wobble embryos. (D) Ratio of junction/cytoplasm signal of F-actin is reduced with p115RhoGEF KD and rescued with p115.S wobble injection. p = <0.0001(***), p = 0.0002(**), and p = 0.0055(*). (E) Ratio of junction/cytoplasm signal of P-MLC is reduced with p115RhoGEF KD and rescued with p115.S wobble injection. p = <0.0001(**). Error bars represent mean ± SEM; significance was calculated using unpaired t tests. Control: n = 10 embryos, 3 experiments; p115.S+L KD: n = 10 embryos, 3 experiments; p115.S KD: n = 10 embryos, 3 experiments; p115.S KD + p115.S wobble: n = 10 embryos, 3 experiments. (F) Sum projection of fixed staining for TJs (anti-ZO-1) in control, p115.S+L KD, p115.S KD, and p115.S KD + p115.S wobble embryos. White dashed rectangle of individual junctions is enlarged below; yellow arrowheads indicate discontinuities in ZO-1 signal. (G) Sum projection of fixed staining for AJs (anti-E-cadherin) in control, p115.S+L KD, p115.S KD, and p115.S KD + p115.S wobble embryos. (H) Ratio of junction/cytoplasm signal of ZO-1 and E-cadherin is reduced with p115RhoGEF KD. Injection of p115.S wobble rescues ZO-1 and E-cadherin signal. For ZO-1 graph: p = 0.0005(**) and p = 0.0019(*). For E-cadherin graph: p = <0.0001(***), p = 0.0013(**), and p = 0.0349(*). Error bars represent mean ± SEM; significance was calculated using unpaired t tests. Control: n = 6 embryos, 2 experiments; p115.S+L KD: n = 6 embryos, 2 experiments; p115.S KD: n = 6 embryos, 2 experiments; p115.S KD + p115.S wobble: n = 6 embryos, 2 experiments.