Journal: bioRxiv
Article Title: Feedback regulation by the RhoA-specific GEF ARHGEF17 regulates actomyosin network disassembly
doi: 10.1101/2024.08.28.610052
Figure Lengend Snippet: A: Fluorescence micrographs of cells stained with Phalloidin (F-actin), Vinculin and pMLC specific antibodies showing the impact of ARHGEF17 KD on cell morphology during spreading. Cells were detached, allowed to spread for 1 hour on fibronectin-coated coverslips. Images acquired using a 100x planApo oil immersion objective. B: Quantification of phalloidin, pMLC and vinculin fluorescence intensities in control and ARHGEF17 KD cells (Images acquired using a 100x objective). Distance intensity plots are shown for intensity of staining from cell border towards cell center. Shaded area indicates standard deviation. n= 477 cells (242 Control, 235 KD). C: Time-lapse series showing cell spreading and polarisation in control versus ARHGEF17 KD cells. Increased spreading and robust contractile F-actin structures are noted in KD cells within the first five hours as compared to control cells. At 12 hours, thicker contractile fibers are observed in ARHGEF17 KD cells. D: Quantitative analysis of area and solidity of control and KD cells after 5 hours of spreading, measured at time point 5 hours. Boxplot with interquartile ranges and superimposed violin plots are shown to provide an intuition about the measurement distributions. White bar indicates population average. n=2033 (945 Control, 1088 KD). Statistical test: Mann-Whitney U test. E: SIM images of spreading cells expressing MLC-mCherry in control and ARHGEF17 KD cells. High magnification panels showing (1) MLC in edge proximal lamella (2) MLC in edge distal lamella. The panels have been oriented to be parallel to the edge, with the retrograde actin flow being oriented from top to bottom (denoted by the red arrow). Scale bars: (A) 10 µm, (C) 5 µm, (E) 10 µm, (E) Insets: 2 µm
Article Snippet: REF-52 cells with optoLARG construct and LifeAct-miRFP were transfected with ARHGEF17-mCherry (kindly provided by Olivier Rocks) and plated on glass bottom well plates (Celvis) which were coated with 5 μg/mL of human plasma fibronectin purified protein (Merck) for one hour at room temperature.
Techniques: Fluorescence, Staining, Control, Standard Deviation, MANN-WHITNEY, Expressing
