Immunofluorescence staining to observe morphological changes after treatment with GSK inhibitors.

<p>(A) Apotome-based microscopic imaging of triple negative MDA-MB-231 cells treated with DMSO (control; panel <i>a</i>), GSK198271A, GSK350559A, GSK494610A, GSK346294A, GSK448459A, GSK237700A, GSK809897X, and GSK1010829B. The samples were pretreated for 72 hours before staining and experiment was performed in triplicate. A phalloidin stain was used to highlight actin filaments (red), DAPI stained for the nucleus (blue). (B) Quantitative morphological analysis of MDA-MB-231 cells after treatment with select GSK compounds. A minimum of 45 cells were quantified per treatment group. Measurements of cellular morphology after immunofluorescence staining and Apotome microscopy imaging are reported. Area is defined as the length of the cell x the width; Circularity = 4(π) x (area/perimeter²). Aspect Ratio = length/width; Nuclear Area Fraction = (Area of Nucleus/Area of Cell). Greater area, cell circularity, aspect ratio and nuclear area fraction are associated with a mesenchymal phenotype due to cytoskeletal rearrangement [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177802#pone.0177802.ref031" target="_blank">31</a>]. For quantification experiments, N ≥ 45, error bars represent SEM and significantly different * p < 0.05, *** p < 0.001.