bsmu FY2021 Annual Report 05

Figure 2. MSA of molecule 3 binding the microtubule array, inhibiting tubulin polymerization regionally triggering prometaphase−metaphase oscillations. (A) Kinetic profiles of tubulin polymerization in the presence or absence of molecule 3 (20 or 100 μM), Taxol (3 μM), and Nocodazole (3 μM). (B) TEM images of the products of tubulin polymerizations. (C) SDS-PAGE and Native-PAGE analysis of supernatant fractions of the products of tubulin polymerization in the absence (MT) or presence of molecule 3 [MT+3]. Tubulin (25 μg) and molecule 3 (100 μM) were applied as loading controls. Immunofluorescence of α-tubulin (D) and β-tubulin (E) in HeLa cells upon the treatment of molecule 3 at 100 μM for 12 h. The nuclei are stained with DAPI. (F) Histogram of the mitotic duration of control HeLa H2B-GFP cells (n > 100). (G) Representative time-lapse microscopy of the mitotic progression of the HeLa H2B-GFP cell (highlighted with pseudo-color). The initiation point was set to 00:00 (hours:minutes). The nuclear envelope breakdown is presented as NEBD. Scale bar represents 5 μm. (H) Histogram of mitosis duration time (gray bars) and cycles of prometaphase−metaphase oscillation (colored bars) of HeLa H2B-GFP cells upon the treatment of molecule 3 (50 μM) (n = 150). (I) Stacked bar graph represents the HeLa cell fate upon the treatment of molecule 3 (50 μM) versus oscillation number. Total 149 cells undergoing prometaphase−metaphase oscillation are counted. (J, K) Representative time-lapse microscopy of the mitotic progression of HeLa H2B-GFP cells upon the treatment of molecule 3 (50 μM). Scale bars represent 5 μm.