PCAIs stimulate MAPK, PI3K/AKT pathways and ROS-Mediated apoptosis in aromatase inhibitor-resistant breast cancer cells while disrupting actin filaments and focal adhesion (IMAGE)
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Figure 10: PCAIs treatment collapses actin filaments and delocalizes focal adhesion proteins. Cells were grown in 8-well ibidi μ-slides overnight and treated with the respective concentrations of PCAIs for 48 h, then fixed and permeabilized. Images of the cells treated with 0 and 5 μM NSL-YHJ-2-27 are shown at higher magnification whereby yellow arrows indicating retracting lamellipodia indicative of collapsing actin filaments. (A) Triplicates of the fixed cells were stained with Alexa Fluor™ 568 Phalloidin for actin filaments. Yellow arrows indicate retracting lamellipodia indicative of the collapsing actin filaments (magnified images). (B) Vinculin punctates were probed using vinculin antibody and visualized using rabbit IgG Alexa Fluor 555 conjugate. White arrows indicate vinculin punctates. (C) Fascin was probed with fascin antibody and visualized using mouse IgG Alexa Fluor 488. White arrows indicate defined fascin spots. Images were captured using Keyence BX-X800 microscope at 40X magnification. (D) Cells treated for 48 h with the indicated concentrations of NSL-YHJ-2-27 or 5 μM of the non-farnesylated PCAIs analog, NSL-YHJ-2-62. Lysis of the cells and analysis by western blotting for vinculin and fascin were conducted as described in the methods. Western blot images and plots of chemiluminescence intensities of the bands following quantification using Image Lab Software normalized against GAPDH against concentration. Data are representative of three independent experiments. Statistical significance (**p < 0.01 in relation to the negative control, 0 μM) was determined by one-way ANOVA with post hoc Dunnett’s test.
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