Figure 3. Loss of AMPK enhances cell adhesion, fibrillogenesis, mechanotransduction, and intracellular stiffness. (A) Adherence of AMPK WT and KO MEFs on fibronectin measured in real-time using the xCELLigence RTCA instrument. Representative curves of cell adhesion over the first 2 h and quantification of relative cell adhesion (cell index) at 2 h after plating are shown. Data are expressed relative to WT and represent means ± SEM. n = 9–11 experiments from four biological repeats. (B) Representative images and quantification of fibronectin fiber length (µm) in AMPK WT and KO MEFs 16 h after supplementation with 10 µg/ml fibronectin. Data are displayed as Tukey box plots. n > 110 fibers from three biological repeats. (A and B) ***, P < 0.0001 (two-tailed Student’s t test). (C) Representative traction force maps and quantification of the mean force (strain energy, pJ) exerted by AMPK WT and KO MEFs plated on fibronectin-coated (5 µg/ml) polyacrylamide gels with a Young’s modulus of ∼1 kPa. Black arrows indicate the direction of traction stress. Cell contours are denoted by white lines. The color code gives the magnitude of traction stress in Pa, which corresponds to forces of pN/µm2. Data are displayed as Tukey box plots. n = 12–14 cells from two biological repeats. *, P = 0.0328 (two-tailed Student’s t test). (D) Viscoelastic relaxation experiment in AMPK WT and KO MEFs. Brightfield image of a KO MEF with an internalized 2-µm diameter microsphere (black arrow) plated on a crossbow-shaped micropattern (dotted lines). The schematic depicts the principle of the experiment. The bead is initially trapped using optical tweezers. A step displacement (Xs = 0.5 µm) is then applied to the cell. The displacement of the bead xb(t) relative to the fixed position of the optical trap is measured. (B–D) Bars: (B) 20 µm; (C and D) 10 µm. (E) Averaged relaxation curves of the bead position after the 0.5-µm step in AMPK WT and KO MEFs. (F–H) Mean values of the rigidity index (F) and of the rheological parameters obtained by fitting the relaxation curves with a SLL model (see the Microrheology in micropatterned cells section of Materials and methods), the intracellular viscosity η (G), and the intracellular spring constant (H) in AMPK WT and KO MEFs. Data shown in E–H are from n = 24 beads from 24 cells from three biological repeats. Error bars represent SEM. **, P = 0.0022 (one-tailed Student’s t test). a.u., arbitrary units.
Figure 3.

Loss of AMPK enhances cell adhesion, fibrillogenesis, mechanotransduction, and intracellular stiffness. (A) Adherence of AMPK WT and KO MEFs on fibronectin measured in real-time using the xCELLigence RTCA instrument. Representative curves of cell adhesion over the first 2 h and quantification of relative cell adhesion (cell index) at 2 h after plating are shown. Data are expressed relative to WT and represent means ± SEM. n = 9–11 experiments from four biological repeats. (B) Representative images and quantification of fibronectin fiber length (µm) in AMPK WT and KO MEFs 16 h after supplementation with 10 µg/ml fibronectin. Data are displayed as Tukey box plots. n > 110 fibers from three biological repeats. (A and B) ***, P < 0.0001 (two-tailed Student’s t test). (C) Representative traction force maps and quantification of the mean force (strain energy, pJ) exerted by AMPK WT and KO MEFs plated on fibronectin-coated (5 µg/ml) polyacrylamide gels with a Young’s modulus of ∼1 kPa. Black arrows indicate the direction of traction stress. Cell contours are denoted by white lines. The color code gives the magnitude of traction stress in Pa, which corresponds to forces of pN/µm2. Data are displayed as Tukey box plots. n = 12–14 cells from two biological repeats. *, P = 0.0328 (two-tailed Student’s t test). (D) Viscoelastic relaxation experiment in AMPK WT and KO MEFs. Brightfield image of a KO MEF with an internalized 2-µm diameter microsphere (black arrow) plated on a crossbow-shaped micropattern (dotted lines). The schematic depicts the principle of the experiment. The bead is initially trapped using optical tweezers. A step displacement (Xs = 0.5 µm) is then applied to the cell. The displacement of the bead xb(t) relative to the fixed position of the optical trap is measured. (B–D) Bars: (B) 20 µm; (C and D) 10 µm. (E) Averaged relaxation curves of the bead position after the 0.5-µm step in AMPK WT and KO MEFs. (F–H) Mean values of the rigidity index (F) and of the rheological parameters obtained by fitting the relaxation curves with a SLL model (see the Microrheology in micropatterned cells section of Materials and methods), the intracellular viscosity η (G), and the intracellular spring constant (H) in AMPK WT and KO MEFs. Data shown in E–H are from n = 24 beads from 24 cells from three biological repeats. Error bars represent SEM. **, P = 0.0022 (one-tailed Student’s t test). a.u., arbitrary units.

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