Supplementary Figure 4: Rnd3 recruits p190B to the ZA when NMII is inhibited.
From: Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions
(a) MCF-7 cells transfected with non-targeting control siRNA or siRNA against Rnd3 (Rnd3 KD) or Rnd1 (Rnd1 KD) and treated with vehicle control DMSO or blebbistatin (BLB; 100 μM, 2 h) were fixed with methanol and stained for p190B Rho GAP and E-cadherin. Representative confocal images were acquired at the apical junctions. (b) Lysates from MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or siRNA targeted against Rnd3 (Rnd3 KD) were immunoblotted for Rnd3 and β-tubulin (loading control). (c) Lysates from MCF-7 cells transfected with Rnd3siRNA along with GFP-tagged Rnd3 constructs (WT, AIIA, S240A, T55V) were immunoblotted for GFP and β-tubulin (loading control). (d) MCF-7 cells transfected with non-targeting siRNA (Ctrl siRNA) or Rnd3 siRNA (Rnd3 KD) and reconstituted with low levels of GFP, or GFP-tagged WT Rnd3 (WT), AIIA Rnd3 (AIIA), S240A Rnd3 (S240A), or T55V Rnd3 (T55V) were treated with either DMSO or blebbistatin (BLB) (100 μM, 2 h), fixed with TCA and stained for RhoA. Asterisks (∗) indicate GFP-tagged Rnd3 expressing cells. (e) MCF-7 cells transfected with GFP-AHPH and non-targeting siRNA (Ctrl siRNA) or Rnd3 siRNA (Rnd3 KD) and reconstituted with low levels of mCherry, or mCherry-tagged WT Rnd3 (WT), AIIA Rnd3 (AIIA), S240A Rnd3 (S240A), T55V Rnd3 (T55V) were treated with either DMSO or blebbistatin (BLB, 100 μM, 2 h) fixed with TCA and stained for GFP. Asterisks (∗) indicate mCherry-tagged Rnd3 expressing cells. Scale bars, 10 μm.
