αV-integrins do not contribute to early adhesion to collagen I in MDCK cells.

<p><b>A</b>) Quantifying early cell adhesion by SCFS. Single control or ItgαV-KD cells were captured onto ConA-coated AFM cantilevers and pressed onto Col I-coated substrates. After the indicated contact times, cells were lifted from the substrate, and the maximum detachment force was recorded as described in materials and methods. More than 100 F–D curves were recorded for each condition. Mean+SD is shown. <b>B</b>) The maximum detachment force between collagen I substrate and control or Itgα2-KD MDCK cells was measured in the absence and presence of antibodies that block β1-integrin adhesion (AIIB2). At least 20 cells were analyzed for each condition. Values show mean ± SD. Membrane tether analysis (see T in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071485#pone.0071485.s003" target="_blank">Fig. S3A</a></b>): <b>C</b>) The distributions of tether rupture forces for control and ItgαV-KD cells both center at a value of ≈92 pN, indicating no differences in cell membrane properties. 78 F–D curves for control and 41 F–D curves for ItgαV-KD cells were analyzed. <b>D</b>) Membrane tether length analysis of control and ItgαV-KD cells to identify possible differences of receptor affinity and cell membrane-cortex interactions. A very similar tether length distribution does not indicate any changes. To assure that analyzed rupture events exclusively originated from membrane tethers force steps within 7 µm after the maximum detachment force were omitted from analysis. <b>E</b>) Control and two independent ItgαV-KD MDCK cell lines were allowed to spread on Col I-coated plastic tissue culture dishes for 75 minutes followed by surface biotinylation. After cell lysis, biotin-bound molecules were precipitated with avidin-beads and relative amounts of β1-and α2-integrins in biotinylated vs. total lysates were determined by SDS-PAGE and western blotting as described in materials and methods. E-Cadherin (E-Cad, asterisk indicates an intracellular precursor of E-Cad) and β-actin were used as biotinylation and loading controls. ns = non-specific. <b>F</b>) Quantification of the relative surface expression of β1- and α2-integrins in the indicated cell lines.