Figure 2.
Figure 2. Expression of mutant (m4) FL-APC leads to reduced F-actin levels in cells without altering MT organization and dynamics or mitochondria distribution. (A) Western blots of whole cell extracts from mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), depleted cells rescued by plasmids expressing si-resistant untagged FL-APC (WT or m4), and cells ectopically expressing untagged FL-APC (WT or m4) probed with APC and tubulin (loading control) antibodies. Below each lane of the blot is the ratio of APC to tubulin relative to control cells. (B) Quantification of APC signal in cells measured by immunofluorescence intensity (n = 30 cells per condition; Fig. S3) and normalized to cell area from cells treated as in A. Data averaged from three or more experiments. Error bars, SD; two-tailed Student’s t test. (C) Representative images showing F-actin organization (rhodamine-phalloidin staining) in mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), and depleted cells rescued by plasmids expressing si-resistant FL-APC (WT or m4). (D) Quantification of mean F-actin density in cells. Data averaged from three independent experiments (n = 60 cells per condition). (E) Representative images showing MT organization (tubulin immunostaining) in mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), and depleted cells rescued by plasmids expressing si-resistant FL-APC (WT or m4). Magnifications are shown for boxed regions. (F) Quantification of MT density at cell periphery normalized to control cells (100%). Data are mean from three independent experiments (n = 50 cells per condition). (D and F) Error bars, SEM; one-way ANOVA Holm-Sidak’s multiple comparison t test. (G) Representative images of GFP-EB1 comets (red) tracked in live cells as in A using TIRF microscopy and analyzed with U-track. Longer red lines reflect faster GFP-EB1 movements. (H) Quantitative analysis of GFP-EB1 comet velocities and lifetimes measured using U-track. Each data point graphed is the mean of ≥3,000 comets tracked during a 5-min observation window in a single cell (n = 10 cells per condition). Error bars, SEM; one-way ANOVA Bonferroni multiple comparison correction test. (I) Representative images of U2OS cells treated as in C and stained with MitoTracker green FM dye. (J) Quantification of mitochondrial dispersal in cells expressed as a ratio of the area of mitochondrial staining to whole cell area. Data averaged from three independent experiments (n = 30 cells per condition). Error bars, SD; one-way ANOVA Bonferroni multiple comparison correction test. Statistical differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant.

Expression of mutant (m4) FL-APC leads to reduced F-actin levels in cells without altering MT organization and dynamics or mitochondria distribution. (A) Western blots of whole cell extracts from mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), depleted cells rescued by plasmids expressing si-resistant untagged FL-APC (WT or m4), and cells ectopically expressing untagged FL-APC (WT or m4) probed with APC and tubulin (loading control) antibodies. Below each lane of the blot is the ratio of APC to tubulin relative to control cells. (B) Quantification of APC signal in cells measured by immunofluorescence intensity (n = 30 cells per condition; Fig. S3) and normalized to cell area from cells treated as in A. Data averaged from three or more experiments. Error bars, SD; two-tailed Student’s t test. (C) Representative images showing F-actin organization (rhodamine-phalloidin staining) in mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), and depleted cells rescued by plasmids expressing si-resistant FL-APC (WT or m4). (D) Quantification of mean F-actin density in cells. Data averaged from three independent experiments (n = 60 cells per condition). (E) Representative images showing MT organization (tubulin immunostaining) in mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), and depleted cells rescued by plasmids expressing si-resistant FL-APC (WT or m4). Magnifications are shown for boxed regions. (F) Quantification of MT density at cell periphery normalized to control cells (100%). Data are mean from three independent experiments (n = 50 cells per condition). (D and F) Error bars, SEM; one-way ANOVA Holm-Sidak’s multiple comparison t test. (G) Representative images of GFP-EB1 comets (red) tracked in live cells as in A using TIRF microscopy and analyzed with U-track. Longer red lines reflect faster GFP-EB1 movements. (H) Quantitative analysis of GFP-EB1 comet velocities and lifetimes measured using U-track. Each data point graphed is the mean of ≥3,000 comets tracked during a 5-min observation window in a single cell (n = 10 cells per condition). Error bars, SEM; one-way ANOVA Bonferroni multiple comparison correction test. (I) Representative images of U2OS cells treated as in C and stained with MitoTracker green FM dye. (J) Quantification of mitochondrial dispersal in cells expressed as a ratio of the area of mitochondrial staining to whole cell area. Data averaged from three independent experiments (n = 30 cells per condition). Error bars, SD; one-way ANOVA Bonferroni multiple comparison correction test. Statistical differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant.

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