Figure S4.
Inhibition of MSCs does not significantly alter the overall morphology of cell–cell junctions. (A) Live imaging of active Rho (mCherry-2xrGBD, magenta) in embryos treated with vehicle (water) or 12.5 µM GsMTx4 (MSC inhibitor). Montage shows that MSC inhibition does not affect Rho activity at the contractile ring, and cells complete cytokinesis successfully. Blue asterisk indicates dividing cells. Time 0 s represents the start of contractile ring formation. (B and B′) Live imaging of active Rho (mCherry-2xrGBD, magenta) and ZO-1 (BFP-ZO-1, blue) in embryos treated with vehicle (water) or 12.5 µM GsMTx4. (B) Quantification shows that MSC inhibition does not significantly affect baseline junctional active RhoA or ZO-1 intensity at apical cell–cell junctions compared with vehicle controls. Junctional intensity of active Rho and ZO-1 from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using Wilcoxon matched-pairs test; n = 30 junctions, 6 experiments. (C–C″) Sum projection of fixed staining for P-MLC (anti-phospho-myosin light chain2, magenta) and F-actin (Alexa Fluor 647 phalloidin, green) in embryos treated with vehicle (water) or 12.5 µM GsMTx4. (C′) Quantification shows that MSC inhibition does not significantly affect the junctional intensity of P-MLC and F-actin at cell–cell junctions compared with vehicle controls. (C″) MSC inhibition reduces the junction/cytoplasm ratio of P-MLC compared with vehicle but does not affect the junction/cytoplasm ratio for F-actin. Data points from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using one-way ANOVA test; n = 45 junctions, 3 experiments. (D) Sum projection of fixed staining for adherens junctions (anti-E-Cadherin, green) and DNA (DAPI, cyan hot) in embryos treated with vehicle (water) or 12.5 µM GsMTx4. (D′) Graph of the normalized intensity of E-Cadherin shows that MSC inhibition does not significantly affect the intensity of E-Cadherin at cell–cell junctions compared with vehicle controls. Data points from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using Mann–Whitney U test; n = 60 junctions, 12 embryos, 3 experiments.

Inhibition of MSCs does not significantly alter the overall morphology of cell–cell junctions. (A) Live imaging of active Rho (mCherry-2xrGBD, magenta) in embryos treated with vehicle (water) or 12.5 µM GsMTx4 (MSC inhibitor). Montage shows that MSC inhibition does not affect Rho activity at the contractile ring, and cells complete cytokinesis successfully. Blue asterisk indicates dividing cells. Time 0 s represents the start of contractile ring formation. (B and B′) Live imaging of active Rho (mCherry-2xrGBD, magenta) and ZO-1 (BFP-ZO-1, blue) in embryos treated with vehicle (water) or 12.5 µM GsMTx4. (B) Quantification shows that MSC inhibition does not significantly affect baseline junctional active RhoA or ZO-1 intensity at apical cell–cell junctions compared with vehicle controls. Junctional intensity of active Rho and ZO-1 from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using Wilcoxon matched-pairs test; n = 30 junctions, 6 experiments. (C–C″) Sum projection of fixed staining for P-MLC (anti-phospho-myosin light chain2, magenta) and F-actin (Alexa Fluor 647 phalloidin, green) in embryos treated with vehicle (water) or 12.5 µM GsMTx4. (C′) Quantification shows that MSC inhibition does not significantly affect the junctional intensity of P-MLC and F-actin at cell–cell junctions compared with vehicle controls. (C″) MSC inhibition reduces the junction/cytoplasm ratio of P-MLC compared with vehicle but does not affect the junction/cytoplasm ratio for F-actin. Data points from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using one-way ANOVA test; n = 45 junctions, 3 experiments. (D) Sum projection of fixed staining for adherens junctions (anti-E-Cadherin, green) and DNA (DAPI, cyan hot) in embryos treated with vehicle (water) or 12.5 µM GsMTx4. (D′) Graph of the normalized intensity of E-Cadherin shows that MSC inhibition does not significantly affect the intensity of E-Cadherin at cell–cell junctions compared with vehicle controls. Data points from paired experiments are color matched. Error bars represent mean ± SEM; significance calculated using Mann–Whitney U test; n = 60 junctions, 12 embryos, 3 experiments.

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