Fig. 6
Knockdown of BRSK2 suppresses AKT, STAT3, and NF-κB signaling, as well as basal autophagy and autophagy induced by nutrient deprivation stress, in breast cancer cells. (A) MDA-MB-231 cells transfected with siControl and validated siRNA targeted to BRSK2 were used for total protein lysate isolation, followed by western blot analysis with antibodies against the indicated proteins, including autophagy-related proteins p62/SQSTM1, ATG3, Beclin-1, active AKT (Ser473), STAT3 (Ser727 and Tyr705), phospho-ERK1/2, and NF-κB (Ser536). An anti-β-actin antibody was used to ensure equal loading and transfer. (B) BRSK2 is knocked down in MDA-MB-231 cells using siRNA targeted to BRSK2, compared to siControl. Knockdown of BRSK2 levels was rescued by ectopic overexpression of BRSK2. Equal amounts of total protein were used for Western blotting with antibodies against LC3, BRSK2, and FLAG-BRSK2. Short and long-exposure BRSK2 western blots were shown. β-Actin was used for equal loading and transfer. (C) BRSK2 protein was knocked down in BT-474 cells using two different siRNAs (siBRSK2 #1 and siBRSK2 #2) compared to siControl. Cells were exposed to ± nutrient-deprived medium for 6 h and subjected to total protein lysate isolation followed by western blotting. (D) BRSK2-knocked-down BT-474 cells with siRNA targeted to BRSK2 (siBRSK2 #1) ± rescued by overexpression of BRSK2 were used for total lysate isolation, followed by western blotting with antibodies against the indicated proteins, including BRSK2 and phospho-AKT. Short and long-exposure BRSK2 western blots were shown. β-Actin was used for equal loading and transfer. All western blotting experiments (n = 3) and representative blots are shown. Blots were scanned, and fold intensities normalized to respective loading control (β Actin) vs. vector control were labeled (ImageJ).
