Figure 5. In cellulo efficacy of GKI-1 in HeLa cells.

A. HeLa cells were transfected with either non-targeting (siSCR) or GWL targeting siRNAs (siGWL). GWL and loading control protein (Tubulin) were detected by western analysis. B., C. HeLa cells treated with siSCR or siGWL (48-h) were arrested in mitosis using nocodazole and treated with DMSO, 25 μM or 50 μM GKI-1. Immunofluorescent staining of p-ENSA (Red) and GWL (Green) was achieved using anti-phospho(Ser67)-ENSA and anti-MASTL(GWL, RIPLY 74C) antibodies and DAPI was used to stain the nucleus (Blue). (C) Quantitation of the IF p-ENSA and GWL signals was performed using the ScanR High-Content Screening Station. The Circularity and Total DAPI parameters were used to identify mitotic cells. D., E. HeLa cells treated with siSCR or siGWL (48-h) were treated with DMSO, 25 or 50 μM GKI-1. 4-h after treatment, μ-slides were mounted onto an Olympus IX73 microscope, within a temperature-controlled 37°C chamber maintained at 5% CO₂, and images were acquired every 5-min for 8.5-h. Time-lapse videos were generated using ImageJ and cellular phenotypes recorded: Mitotic events (D), mitotic arrest (E, F) and failed cytokinesis G. F. Kymographs showing an example of typical phenotypes were generated by capturing images every 5 minutes for 80 minutes. Image sequences were chosen to start just before mitotic entry. (B - G) A total of 3 - 5 biological replicates were completed per condition and the t-test statistical module of Prism 6.0 was used to determine p-values (ns (not statistically significant): P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001).