Panel A shows fluorescence images of Paxillin in cells cultured on Fibronectin or Poly L Lysine for 1 hour, 4 hours, and 24 hours, with enlarged views of selected adhesion regions. Panel B shows a superplot of the number of CARTS per cell, with the x-axis labeled 1 hour, 4 hours, 24 hours for Trypsin and Ethylenediaminetetraacetic acid, and the y-axis labeled Number of CARTS per cell. Panel C shows fluorescence images of Vinculin in cells cultured on 2 kilopascal and 30 kilopascal substrates. Panel D shows fluorescence images of Active beta 1 integrin (9EG7), Paxillin, and merged images in cells cultured on 2 kilopascal, 30 kilopascal, and Glass substrates. Panel E shows a superplot of Active beta 1 signal per cell (arbitrary units), with the x-axis labeled 2 kilopascal, 30 kilopascal, Glass and the y-axis labeled Active beta 1 signal per cell (arbitrary units). Panel F shows a superplot of focal adhesion area per cell (square micrometers), with the x-axis labeled 2 kilopascal, 30 kilopascal, Glass and the y-axis labeled FA area per cell (square micrometers).
Adhesion-dependent cell states associate with CARTS formation. (A) Confocal fluorescence microscopy images of fixed HeLa cells seeded on FN or PLL for the indicated time points and immunostained against endogenous paxillin. Zoom-ins of the highlighted regions are shown on top. Scale bar, 10 µm. (B) SuperPlot showing the number of CARTS per cell (small, light-colored symbols; N∼10 per biological replicate) on cells lifted using trypsin or EDTA (see Materials and methods). The mean value for each independent biological replicate (larger, black-outlined circles; n = 3). A repeated-measures two-way ANOVA test was performed, and P values were obtained using Tukey’s post hoc multiple comparison test, with only the values for the comparisons between trypsin and EDTA being shown. (C) Spinning disk confocal fluorescence microscopy images of fixed HeLa cells that were plated on stiff (30 kPa) and soft (2 kPa) PAA gels, let spread for 4 h, fixed, and immunostained for vinculin, a FA marker. Scale bars, 10 µm. (D) Confocal microscopy images of fixed HeLa cells plated on FN-coated glass coverslips, or on stiff (30 kPa) or soft (2 kPa) FN-coated PAA gels. 4 h after plating them, cells were fixed and processed for immunofluorescence microscopy against endogenous active β1 integrin (9EG7 antibody) and paxillin. (E and F) SuperPlots showing quantification of the fluorescence intensity signal of active β1 integrin per cell (E) and of the FA area per cell (F) from cells in D. A repeated-measures one-way ANOVA test was performed using Tukey’s post hoc multiple comparison test (n = 3 replicates, N∼10 cells per replicate).