Fig. 4

MT acetylation is required to induce PPLL extensions. a Luc- and Mec-17-depleted D723H cells were spread 48 h post siRNA (50 nM) on fibrinogen for 2 or 5 h. Cell extracts were analyzed by Western blot. Mec-17 depletion correlates with loss of Ac-Tub, while PolyE-Tub and total tubulin are not affected. Representative images (MIP) of luc- and Mec-17-depleted D723H cells stained as indicated. Quantification of Ac-MT/Total MT ratio shows the loss of Ac-Tub staining in MEC-17-depleted cells (scatter dot plot shows mean ± SEM; n = 3 at least 100 cells per condition were analyzed. Unpaired student t test, two-tailed ***P < 0.0001). Loss of Ac-Tubulin in Mec-17-depleted cells correlates with acquisition of a cell square shape phenotype. Bar is 30 μm. b D723H cells treated as in A, but using Mec-17 siRNA 15 nM. Carrier or TSA was added 30 min after cell spreading, and cells were analyzed 4 h later. Western blot shows that MEC-17 depletion is partial under these conditions. TSA treatment allows stabilization of Ac-MTs. MIP of representative images of luc-, Mec17, and Mec-17+TSA-depleted D723H cells stained for total and Ac-MTs. Bar is 20 μm. Rescue of PPLL elongation is quantified using the AR aspect ratio function (particle analysis) of ImageJ software that measures the ratio between the cell major and minor axis. (n = 3 at least 100 cells per condition were analyzed; graph, left panel: mean AR ratio, unpaired Student t test, two-tailed ***P < 0.0001; right panel graph: percentage of cells with AR ratio above or under 2.5 value, error bars are SEM