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. 2019 Mar 22;38(11):e99630. doi: 10.15252/embj.201899630

Figure 3. Assembly of microtubules and F‐actin on isolated centrosomes.

Figure 3

  1. Two sets of representative images showing fluorescent microtubules and F‐actin assembled from isolated centrosomes. Centrosomes were isolated from Jurkat cells expressing centrin1‐GFP. Upper and lower lines show F‐actin and microtubules radiating from two distinct centrosomes with low (top) and high (bottom) densities of F‐actin. Scale bars: 10 μm.
  2. The graph shows the number of microtubules per centrosome relative to the density of actin filaments. Inset shows F‐actin at the centrosome with a FIRE look‐up table and a 20‐μm‐wide circle in which F‐actin fluorescence intensity is measured. Measurements were pooled from five independent experiments; n = 50.
  3. Microtubules (top line) and F‐actin (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm.
  4. The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin. Data were pooled from two independent experiments; 0 μM: n = 21; 0.3 μM: n = 17; 0.5 μM: n = 17; 1.0 μM: n = 17. ****P < 0.001 Mann–Whitney test.
  5. The image shows the density of F‐actin (in the presence of 1 μM of actin monomers) at the centrosome colour‐coded with the FIRE look‐up table and the definition of central, peripheral and distal regions corresponding to decreasing concentrations of F‐actin. Scale bar: 20 μm. The graph shows the measurements of microtubule growth rate in each region. Data were pooled from three independent experiments: central zone: n = 58, peripheral: n = 104, outside: n = 61; n. s. means no statistical difference between the data set according to Mann–Whitney test.
  6. The graphs show the various intensities of centrosome immuno‐staining with antibodies against gamma‐tubulin on the same coverslip depending on the presence/absence of F‐actin (left) or on the amount of F‐actin (right). Data were pooled from three independent experiments. Left graph: without F‐actin n = 69, with F‐actin n = 26, right graph n = 26. n.s. means no statistical difference between the data set according to Mann–Whitney test.