Figure 8.
Figure 8. Inhibition of CKI suppresses microtubule growth in epithelial cells. (A, left) Cytoplasmic cell extracts (CCE) of RPE1 cells were processed for immunoprecipitation (ip) with random IgG (control [con]) or anti-p150glued (p150glued) antibodies. (right) Cytoplasmic cell extracts of RPE1 cells were subjected to pull-down assays with GST, GST-EB1 (EB1), or GST-EB3 (EB3) and were immunoblotted with antibodies as indicated. Recombinant GST products are visible in Ponceau S staining below. WB, Western blotting. (B) Analysis of EB3-GFP comets in Jurkat cells treated with DMSO or 100 µM D4476. The box plot shows the distribution of mean growth speeds of tracked EB3-GFP comets per cell. (C) Microtubules were depolymerized on ice in DMSO- or 50 µM D447-treated RPE1 cells. Cells were then shifted to 37°C for 10 min to allow microtubule polymerization. Cells are stained with anti–α-tubulin antibodies. Bar, 10 µm. (D) Number of EB3-GFP growth tracks in DMSO- or 25 µM D4476-treated RPE1 cells. The box plot shows the number of EB3-GFP growth tracks per cell. (E) Analysis of EB3-GFP comets in RPE1 cells treated with DMSO or 25 µM D4476. (left) The box plot shows distribution of mean growth speeds of tracked EB3-GFP comets per cell. (right) The table lists microtubule dynamic parameters by plusTipTracker. Pshrink indicates the probability of shrinkage at the end of grouped growth tracks. Pgrowth and Pfgap indicate the percentage of time all microtubules spend in growth or in pause between growth phases, respectively. **, P < 0.0001; #, P = 0.05 (using permutation t tests). In box plots, whiskers are set at 5–95 percentiles; horizontal lines mark the median, and boxes indicate interquartile range (25–75%).

Inhibition of CKI suppresses microtubule growth in epithelial cells. (A, left) Cytoplasmic cell extracts (CCE) of RPE1 cells were processed for immunoprecipitation (ip) with random IgG (control [con]) or anti-p150glued (p150glued) antibodies. (right) Cytoplasmic cell extracts of RPE1 cells were subjected to pull-down assays with GST, GST-EB1 (EB1), or GST-EB3 (EB3) and were immunoblotted with antibodies as indicated. Recombinant GST products are visible in Ponceau S staining below. WB, Western blotting. (B) Analysis of EB3-GFP comets in Jurkat cells treated with DMSO or 100 µM D4476. The box plot shows the distribution of mean growth speeds of tracked EB3-GFP comets per cell. (C) Microtubules were depolymerized on ice in DMSO- or 50 µM D447-treated RPE1 cells. Cells were then shifted to 37°C for 10 min to allow microtubule polymerization. Cells are stained with anti–α-tubulin antibodies. Bar, 10 µm. (D) Number of EB3-GFP growth tracks in DMSO- or 25 µM D4476-treated RPE1 cells. The box plot shows the number of EB3-GFP growth tracks per cell. (E) Analysis of EB3-GFP comets in RPE1 cells treated with DMSO or 25 µM D4476. (left) The box plot shows distribution of mean growth speeds of tracked EB3-GFP comets per cell. (right) The table lists microtubule dynamic parameters by plusTipTracker. Pshrink indicates the probability of shrinkage at the end of grouped growth tracks. Pgrowth and Pfgap indicate the percentage of time all microtubules spend in growth or in pause between growth phases, respectively. **, P < 0.0001; #, P = 0.05 (using permutation t tests). In box plots, whiskers are set at 5–95 percentiles; horizontal lines mark the median, and boxes indicate interquartile range (25–75%).

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