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. 2017 Nov 6;216(11):3713–3728. doi: 10.1083/jcb.201610039

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© 2017 Pitaval et al.

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Figure 4. — Microtubule stabilization after serum starvation promotes centrosome migration. (A) Identification of cold-resistant microtubules. Serum-starved RPE1 cells were subjected to cold shock (on ice for 12 min) and fixed and stained with an antibody to α-tubulin (lookup table [LUT] fire [ImageJ, National Institutes of Health]). Images show five examples of serum-starved and five examples of serum-fed cells (LUT fire). The graph shows measurements of α-tubulin fluorescence intensity after cold shock for various delays after serum removal (results of three independent experiments, n = 125 cells per condition). (B) 3D numerical simulations of microtubule growth from the centrosome at the basal pole. They showed that longer microtubules, assembled by reducing the catastrophe rate, induced a symmetry break in the network architecture that was capable of pushing the centrosome to the apical surface. The graph shows the centrosome z position according to the catastrophe rate. (C and D) Identification of cold-resistant microtubules in serum-fed cells treated with either control siRNA or siRNA against the tubulin sequestering protein stathmin 1. The same conditions as in A. Images show five examples of serum-fed cells treated with control siRNA and five examples of serum-fed cells treated with stathmin 1 siRNA (C). (D) Graph shows measurements of α-tubulin fluorescence intensity after cold shock (results of two independent experiments, control siRNA, n = 125 cells; stathmin 1 siRNA, two siRNA sequences, n = 125 cells each). Horizontal bars show mean values. (E) Stathmin 1 was depleted by siRNA from RPE1 cells cultured in the presence of serum to promote microtubule growth and observe its effect on centrosome position. Cells were fixed and stained for α-tubulin, γ-tubulin, and DNA. Centrosomes are indicated by white arrowheads, and the microtubule bundle is shown with a yellow arrow (left side view images). The graph shows cell percentage displaying basal centrosome (located from 0 to 2 µm above the glass substrate), intermediate centrosome (between 2 and 3 µm above the glass substrate), and apical centrosome (located >3 µm above the glass substrate; results of three independent experiments, control siRNA, n = 100 cells; stathmin 1 siRNA, two siRNA sequences, n = 150 cells each). (F) Time-lapse imaging for 80 min of serum-fed RPE1 cells expressing EGFP-centrin1 treated with control siRNA or siRNA against stathmin 1, then fixation/immunostaining for α-tubulin. The left graph shows the percentage of cells exhibiting a moving centrosome toward the apical pole in each condition (siCTR vs. siStathmin 1). The right graph shows microtubule bundle orientation frequency for each condition (one experiment, control siRNA, n = 63 cells; siRNA against stathmin 1, n = 45 cells). Bars: (x and y) 10 µm; (z) 2.5 µm. a.u., arbitrary units. ****, P < 0.0001.