Fig. 8
Suppression of BRSK2 expression or inhibition by GW296115 reduces breast cancer cell growth and autophagy, thereby enhancing their apoptosis induced by nutrient deprivation stress. BT-474 (A) and MDA-MB-231 (B) cells were transfected with siRNA (Dharmacon, #1) targeted to BRSK2 or control siRNA for 48 h, and cells were exposed to ± nutrient-deprived medium for another 6 h, followed by total protein lysate isolation for western blotting analysis. BRSK2 downregulation efficiencies were validated by western blotting with anti-BRSK2 antibodies. Autophagy marker protein antibodies (anti-LC3) and apoptotic marker protein antibodies (anti-cleaved PARP, anti-cleaved caspase-7, and anti-cleaved caspase-9) were used, along with β-actin for equal loading. Proteins from duplicate cultures were resolved by SDS-PAGE, and cytochrome c released from mitochondria into the cytosol was assessed by immunoblotting using antibodies against cytochrome c (Cyc-c) and β-tubulin to ensure equal loading and transfer. Similar results were obtained in three additional experiments. All the apoptosis Western blots were also confirmed with siBRSK2 #2 (Qiagen, not shown). (C) Pharmacological inhibition of BRSK2, using GW296115 (2–5 µM) versus vehicle, decreased LC3-I/II protein levels but enhanced cleaved caspase. 7 and PARP proteins, as determined by western blotting in MDA-MB-231 cells. β-Actin was used as a loading control and for equal transfer. N = 3, representative blots were shown—similar data obtained in other breast cancer cells. All the blots were scanned, and fold intensities normalized to respective loading control vs. vector control were labeled (ImageJ). (D) Inhibition of BRSK2 kinase via GW296115 (2 µM) for 24 h reduced MDA-MB-231 cell growth, n = 5, Student’s t-test, ***p < 0.001(GW296115 vs. vehicle control). (E) GW296115 (2–3 µM) vs. vehicle treatment for 72 h significantly reduced 3D Matrigel tumor cell invasiveness (invadopodia) in MDA-MB-231 cells. Representative light microscopic images (n = 10), scale bar 200 µm, quantification of invadopodia/spheroid, n = 10, ANOVA and post-hoc t-test, ***p < 0.001 vs. vehicle.
