Fig. 4: Maspin regulates microtubule dynamics in vitro and in cells.

a Western blot analysis for Maspin in MCF-10A WT, KO C10 and KO D9 cells stably expressing EB1-mCherry. Cells were transiently transfected with GFP-Maspin or the control GFP-only for 24 h. Cell lysates were sequentially probed using an anti-GFP antibody followed by an anti-Maspin antibody. Anti-GAPDH antibody was used as a loading control; (b) Live imaging of MCF-10A WT, KO C10 and KO D9 cells stably expressing EB1-mCherry and transiently expressing GFP-Maspin or the control GFP-only alone; (c) Microtubule mean growth rates (µm/min) in WT (n = 148 comets, 11 cells), KO C10 (n = 135 comets, 9 cells), KO C10 + GFP-only (n = 100 comets, 8 cells), KO C10 + GFP-Maspin (n = 146 comets, 14 cells), KO D9 (n = 142 comets, 7 cells), KO D9 + GFP-only (n = 110 comets, 9 cells) and KO D9 + GFP-Maspin (n = 119 comets, 10 cells); (d) Schematic representation of dynamic microtubules using an in vitro reconstitution assay (upper). Pre-assembled TAMRA-labeled GMPCPP-seeds in the presence of Atto633-tubulin and purified GFP-Maspin at different concentrations were visualized by TIRF microscopy (lower). Scale bar: 10 µm; (e) Representative kymographs extracted from dynamic microtubules in (d). Scale bar: 5 µm (horizontal), time: 5 min (vertical); (f) Plot of microtubule growth rates as a function of GFP-Maspin concentration with 11 µM tubulin. Control (0 nM; n = 20 microtubules), GFP-Maspin (50 nM; n = 15 microtubules) and GFP-Maspin (500 nM; n = 17 microtubules); (g) Fluorescence intensity over time for GMPCPP-seeds exposed to GFP-Maspin (500 nM). Maspin binds to the seeds and tubulin is increasingly recruited to them (black arrows, (d)).