Figure 2. APC-m4 alters actin dynamics... | Rockefeller University Press

Figure 2.

$Figure 2. APC-m4 alters actin dynamics at FAs. U2OS cells were depleted of endogenous APC and rescued with refractory APC constructs (APC-WT or APC-m4) along with plasmids expressing GFP-actin and mCherry-zyxin. (A) FRAP analysis, in which ROI were selected where GFP-actin and mCherry-zyxin signals overlap (see orange box in cartoon). ROIs were then bleached and monitored for GFP-actin fluorescence recovery. Graphs show mean recovery profiles normalized to zero after bleaching. Data averaged from three independent experiments (n = 30 ROIs from n = 15 cells per condition). (B) FRAP experiments as in A except that ROIs were selected along stress fibers at a distance (>5 µm) from FAs (see orange box in cartoon). Graphs show mean recovery profiles normalized to zero after bleaching. Data averaged from three independent experiments (n = 30 ROIs from n = 15 cells per condition). (C) Average time to 50% maximal recovery for experiments in A. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (D) Average immobile fraction (does not recover in observation window) for experiments in A. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: *, P < 0.05. (E) Average time to 50% maximal recovery for experiments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (F) Average immobile fraction for experiments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant.$

APC-m4 alters actin dynamics at FAs. U2OS cells were depleted of endogenous APC and rescued with refractory APC constructs (APC-WT or APC-m4) along with plasmids expressing GFP-actin and mCherry-zyxin. (A) FRAP analysis, in which ROI were selected where GFP-actin and mCherry-zyxin signals overlap (see orange box in cartoon). ROIs were then bleached and monitored for GFP-actin fluorescence recovery. Graphs show mean recovery profiles normalized to zero after bleaching. Data averaged from three independent experiments (n = 30 ROIs from n = 15 cells per condition). (B) FRAP experiments as in A except that ROIs were selected along stress fibers at a distance (>5 µm) from FAs (see orange box in cartoon). Graphs show mean recovery profiles normalized to zero after bleaching. Data averaged from three independent experiments (n = 30 ROIs from n = 15 cells per condition). (C) Average time to 50% maximal recovery for experiments in A. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (D) Average immobile fraction (does not recover in observation window) for experiments in A. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: *, P < 0.05. (E) Average time to 50% maximal recovery for experiments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (F) Average immobile fraction for experiments in B. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant.