α-Tubulin immunofluorescence and microtubules analysis

For cells imaging 65.000 BMDMs were seeded into each well of a Nunc™ Lab-Tek™ II Chamber Slide™ System (Thermo Fisher Scientific, USA) maintaining culturing conditions, as described above. After these treatments media was removed and cells were washed in PBS (Thermo Fisher Scientific, USA). Fixation was performed using a 3.7% solution of paraformaldehyde (Sigma Aldrich, USA) for 10 minutes; 3 washes were performed after cell fixation. Images of microtubules architecture were obtained by immunostaining; α-tubulin antibody (Sigma – T5168) was used as primary antibody and Abcam – ab6786 was used as secondary antibody according to vendors indications. A 100× objective was used and a z-stack series was performed (≥21 steps of 300 nm each were acquired per image). Images were acquired by using an A1-Nikon confocal microscope (Nikon Corporation, Japan). For both analyses, nuclei were stained using DAPI (Thermo Fisher Scientific, USA) following vendors indications. For the analysis of the recovery of microtubules structure and the restored internalization capability, the media was removed after the overnight treatment with MPN, 1 wash with PBS was performer and medium was replaced.

For α-tubulin analysis, the following procedure was followed: (i) for each of the images composing a z-stack series, a binary 2D map of α-tubulin was generated (sample images are provided in Fig. S16–S18, ESI^†). (ii) 2D binary maps from the same z-stack series were used to generate a binary 3D α-tubulin map (Fig. 4d). (iii) Binary 3D α-tubulin map were used to find α-tubulin branches (Fig. 4e) (movies report the z-stack reconstruction of the cells, ESI^†). Following these analyses, it was possible to quantify the total microtubules extension and the number of microtubules branches inside each of the cells and for the 3 different conditions (both measures were normalized over cell extension) (n ≥ 10). For the generation of 2D α-tubulin map same threshold was selected for all the focal planes, the binary image was processed to define the microtubules using NIS-Software (Nikon Corporation, Japan). The normalization over cell extension was performed by selecting a permissive threshold (highlighting the whole cell body) on tubulin signal on a maximum intensity projection image generated by each of the z-stack series. Normalization on cell extension was preferred over normalization on cell number due to the difference in cell extension among cells. Statistical analyses were performed using ANOVA. Data are presented as means ± SD.