αV-Integrins Are Required for Mechanotransduction in MDCK Epithelial Cells
Figure 6
Mechanotransduction processes are abrogated in ItgαV-KD MDCK cells.
A) Control and ItgαV-KD MDCK cells were trypsinized and seeded onto stiff (10% PAA) or soft (3% PAA) Col I-coated substrates. Cells were allowed to spread overnight followed by PFA-fixation and imaged using DIC-microscopy. Scale bars are 20 µm. B) Determining the cell elasticity of control and ItgαV-KD cells. Cells were seeded onto Col I-coated substrate and allowed to bind and spread for 12 hours. Cell elasticity was probed with 5 or 20 µm glass beads attached to an AFM cantilever. More than 60 cells per cell type and bead size were probed. Mean+SD is shown. P-values <0.01 are signified by (*). C) Control and ItgαV-KD MDCK cells growing on Col I-coated substrate were probed with a 5 µm bead attached to a tipless cantilever (Figure S3B). After making initial contact between Col I-coated bead and cell with a contact force of 1 nN for 120 seconds, the cantilever was oscillated at a frequency of 0.25 Hz and amplitude of 200 nm for 20 minutes. During oscillation, the force acting on the cantilever was recorded. More than 20 control and ItgαV-KD cells were analyzed. D) The indicated amounts of control and two independent talin (Tln)-1-KD cell line lysates were loaded for SDS-PAGE followed by western blotting with mouse anti-talin antibodies. E) Thirty micrograms of total lysates from control and two independent FAK-KD cell lines were loaded for SDS-PAGE followed by western blotting with mouse anti-FAK antibodies. F) Control and the indicated KD cell lines were trypsinized and seeded onto stiff (10% PAA) or soft (3% PAA) Col I-coated substrates. Cells were allowed to spread overnight followed by PFA-fixation and imaged using DIC-microscopy. Scale bars are 20 µm.