Additional file 10: of Microtubule polyglutamylation and acetylation drive microtubule dynamics critical for platelet formation

Figure S3. Increasing MT acetylation in WT cells is not sufficient to induce PPLL elongations. (A) WT cells treated with low TSA drug concentrations (ng/ml) and spread for 16 h on fibrinogen were analyzed for increased total acetylation level by western blot and for induction of PPLL extensions by immunofluorescence. Representative low magnification and wide field acquired tubulin stained images are shown to visualize cell shape (Bar, 150 μm). Quantification of the AR ratio: n = 3, at least 200 cells per condition were analyzed. Graph shows mean AR ratio, unpaired student t-test, Two tailed ***P < 0.0001. Error bars are SEM (B-C) Modulation contrast time lapse. Cell edges were enhanced using the ImageJ software process enhance contrast function (8%). (B) D723H cells treated with carrier, TBSA (10 μM) or TSA (100 ng/ml) were analyzed for increased Ac-MTs level by Western blot (6 h drug treatment) and for PPLL behavior by time lapse. Bar 50 μm. In drug treated cells, induced PPLL tend to retract and widen with time. (C) Mouse liver-derived-megakaryocytes spread on fibrinogen are treated with carrier or TBSA. Bar 80 μm. As observed on D723H cells, TBSA treatment induced retraction and widening of induced proplatelet (n = 2, 15 megakaryocytes). For better visualization please see corresponding movies. (PDF 9633 kb