*2.12. Quantification of Microtubule Density*

For the quantitative analysis of the microtubule density, we analysed the fluorescence images obtained by staining the sample with Hoechst (blue) and αTubulin (green). Excitation/emission wavelengths used were 346/460 nm (blue) and 488/520 nm (green). At least 500 cells were analysed for each sample. For quantification analysis, we firstly removed the background to highlight the strong signal from tubulin by applying a Gaussian filter to images, and then we subtracted this image from the original one. Afterwards, we applied a threshold to segmen<sup>t</sup> the tubulin and create a binary image of these structures. Finally, we compared this mask to the mask of the whole area in the image field, which is covered by cell. Thus, to obtain a quantification of tubulin density, we divided these two values, obtaining the fraction of pixels, which appears to be crossed by tubulin structures.
