Figure 4.
Figure 4. Mitotic defects after GTSE1 depletion are dependent on the activity of the MT depolymerase MCAK. (A) MCAK coimmunoprecipitates with GTSE1. Western blot after immunoprecipitation (IP) from U2OS cell lysates using either anti-GTSE1 or anti-GFP antibodies and probing with anti-GTSE1 or anti-MCAK antibodies. (B) Western blot after immunoprecipitation from U2OS NFLAP-GTSE1 cell lysates using either anti-GFP or anti–c-myc antibodies and probing with anti-GFP or anti-MCAK antibodies. (C) Immunofluorescence images of mitotic U2OS cells after control, GTSE1, MCAK, or combined GTSE1 and MCAK RNAi, stained for DNA (DAPI) and MTs (tubulin). (D) Quantification of the mean length of astral MTs in three dimensions from immunofluorescence analysis as shown in C. 10 astral MTs were measured per cell. n = 10 cells per experiment; 3 experiments per condition. (E) Quantification of the percentage of cells lacking astral MTs from immunofluorescence analysis as shown in C. n > 100 cells per experiment; 3 experiments per condition. (F) Quantification of inner spindle tubulin fluorescence intensity from immunofluorescence analysis as shown in C. n ≥ 19 per experiment; 3 experiments per condition. (G) Analysis of spindle orientation. Quantification of the percentage of metaphase cells with a spindle tilt angle of >20° is shown for each RNAi condition. n > 140 cells; 4 experiments per condition. (H) Quantification of the percentage of cells with misaligned chromosomes from immunofluorescence analysis as shown in C. n > 100 cells per experiments; 3 experiments per condition. (I) Immunofluorescence images of mitotic U2OS cells showing MCAK localization after control and GTSE1 RNAi. Cells were stained for MCAK, GTSE1, and MTs (tubulin). (J) Quantification of MCAK intensity on the inner spindle normalized to tubulin intensity in U2OS cells using immunofluorescence images after control and GTSE1 RNAi. n ≥ 105 cells; 3 independent experiments. P-values were obtained using a Mann-Whitney U test. (K) Quantification of MCAK intensity using GFP fluorescence on the inner spindle normalized to tubulin intensity in HeLa cells expressing BAC-based MCAK-GFP using immunofluorescence images after control and GTSE1 RNAi. n ≥ 89 cells; 3 independent experiments. P-values were obtained using a Mann-Whitney U test. Bars, 5 µm. All error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Mitotic defects after GTSE1 depletion are dependent on the activity of the MT depolymerase MCAK. (A) MCAK coimmunoprecipitates with GTSE1. Western blot after immunoprecipitation (IP) from U2OS cell lysates using either anti-GTSE1 or anti-GFP antibodies and probing with anti-GTSE1 or anti-MCAK antibodies. (B) Western blot after immunoprecipitation from U2OS NFLAP-GTSE1 cell lysates using either anti-GFP or anti–c-myc antibodies and probing with anti-GFP or anti-MCAK antibodies. (C) Immunofluorescence images of mitotic U2OS cells after control, GTSE1, MCAK, or combined GTSE1 and MCAK RNAi, stained for DNA (DAPI) and MTs (tubulin). (D) Quantification of the mean length of astral MTs in three dimensions from immunofluorescence analysis as shown in C. 10 astral MTs were measured per cell. n = 10 cells per experiment; 3 experiments per condition. (E) Quantification of the percentage of cells lacking astral MTs from immunofluorescence analysis as shown in C. n > 100 cells per experiment; 3 experiments per condition. (F) Quantification of inner spindle tubulin fluorescence intensity from immunofluorescence analysis as shown in C. n ≥ 19 per experiment; 3 experiments per condition. (G) Analysis of spindle orientation. Quantification of the percentage of metaphase cells with a spindle tilt angle of >20° is shown for each RNAi condition. n > 140 cells; 4 experiments per condition. (H) Quantification of the percentage of cells with misaligned chromosomes from immunofluorescence analysis as shown in C. n > 100 cells per experiments; 3 experiments per condition. (I) Immunofluorescence images of mitotic U2OS cells showing MCAK localization after control and GTSE1 RNAi. Cells were stained for MCAK, GTSE1, and MTs (tubulin). (J) Quantification of MCAK intensity on the inner spindle normalized to tubulin intensity in U2OS cells using immunofluorescence images after control and GTSE1 RNAi. n ≥ 105 cells; 3 independent experiments. P-values were obtained using a Mann-Whitney U test. (K) Quantification of MCAK intensity using GFP fluorescence on the inner spindle normalized to tubulin intensity in HeLa cells expressing BAC-based MCAK-GFP using immunofluorescence images after control and GTSE1 RNAi. n ≥ 89 cells; 3 independent experiments. P-values were obtained using a Mann-Whitney U test. Bars, 5 µm. All error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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