Figure 4.
Figure 4. Expression of mutant (m4) FL-APC disrupts FA turnover. (A) Representative images showing GFP–FL-APC (WT and m4) colocalization with mCherry-Zyxin in U2OS cell. Graph on the right shows Mander’s correlation coefficient (M1 and M2) value of overlap between GFP-APC and mCherry-Zyxin signals (n = 35 cells per condition). (B) Western blots of whole cell extracts from U2OS cells depleted of endogenous APC (si-APC) and rescued by plasmids expressing si-resistant GFP-tagged FL-APC (WT or m4), and cells ectopically expressing GFP-tagged FL-APC (WT or m4) probed with antibodies to GFP, APC, and tubulin (loading control). (C) Western blot analysis of phospho-Paxillin and tubulin (loading control) levels. Lysates are from mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), depleted cells rescued by plasmids expressing si-resistant FL-APC constructs (rescue FL-WT and FL-m4), and nondepleted cells expressing FL-APC constructs (ectopic FL-WT and FL-m4). Data averaged from three separate blots. Error bars, SD; two-tailed Student’s t test. (D) Representative images of cells as in A stained with phospho-Paxillin antibodies. Magnifications correspond to boxed regions. (E) Quantification of FA size (surface area) measured by phospho-Paxillin immunostaining as in D. Data averaged from three independent experiments (n = 30 FAs per condition). (F) Quantification of FA density at the cell periphery in boxed (10 × 10 µm) regions. Data averaged from three independent experiments (n = 20 boxes per condition). (E and F) Error bars, SEM; one-way ANOVA Holm-Sidak’s multiple comparison test. (G) Representative images of cells treated as in D stained with antibodies against phospho-Paxillin (gray) or tubulin (magenta). Merge panels show overlap of signals, and magnification is from boxed regions. (H) Time-lapse TIRF imaging of FAs marked by GFP-Paxillin in cells treated as in A. Images show overlay of GFP-Paxillin signal at 0 min and 20 min, with two representative magnifications from boxed regions per condition. (I) Thresholded-Mander’s correlation coefficient (tM1 and tM2) value of GFP-Paxillin signal overlap at 0 and 20 min from 10 cells as in H. Significant differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant.

Expression of mutant (m4) FL-APC disrupts FA turnover. (A) Representative images showing GFP–FL-APC (WT and m4) colocalization with mCherry-Zyxin in U2OS cell. Graph on the right shows Mander’s correlation coefficient (M1 and M2) value of overlap between GFP-APC and mCherry-Zyxin signals (n = 35 cells per condition). (B) Western blots of whole cell extracts from U2OS cells depleted of endogenous APC (si-APC) and rescued by plasmids expressing si-resistant GFP-tagged FL-APC (WT or m4), and cells ectopically expressing GFP-tagged FL-APC (WT or m4) probed with antibodies to GFP, APC, and tubulin (loading control). (C) Western blot analysis of phospho-Paxillin and tubulin (loading control) levels. Lysates are from mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), depleted cells rescued by plasmids expressing si-resistant FL-APC constructs (rescue FL-WT and FL-m4), and nondepleted cells expressing FL-APC constructs (ectopic FL-WT and FL-m4). Data averaged from three separate blots. Error bars, SD; two-tailed Student’s t test. (D) Representative images of cells as in A stained with phospho-Paxillin antibodies. Magnifications correspond to boxed regions. (E) Quantification of FA size (surface area) measured by phospho-Paxillin immunostaining as in D. Data averaged from three independent experiments (n = 30 FAs per condition). (F) Quantification of FA density at the cell periphery in boxed (10 × 10 µm) regions. Data averaged from three independent experiments (n = 20 boxes per condition). (E and F) Error bars, SEM; one-way ANOVA Holm-Sidak’s multiple comparison test. (G) Representative images of cells treated as in D stained with antibodies against phospho-Paxillin (gray) or tubulin (magenta). Merge panels show overlap of signals, and magnification is from boxed regions. (H) Time-lapse TIRF imaging of FAs marked by GFP-Paxillin in cells treated as in A. Images show overlay of GFP-Paxillin signal at 0 min and 20 min, with two representative magnifications from boxed regions per condition. (I) Thresholded-Mander’s correlation coefficient (tM1 and tM2) value of GFP-Paxillin signal overlap at 0 and 20 min from 10 cells as in H. Significant differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant.

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