Fig. 3
BRSK2 ectopic expression in breast cancer cells is associated with tumor cell survival pathways linked to AKT and activation of master transcription factors STAT3 and NF-κB. BT-474 (A), MDA-MB-231 (B), and MDA-MB-231/BR (C) cells were transfected with the empty vector, BRSK2, or the BRSK1 plasmids for 48 h, and total protein lysates were prepared for western blot analysis with the indicated antibodies. For some experiments, 3D cell cultures were established 24 h after plasmid transfection. (D) MDA-MB-231/BR cells, as shown, were grown for an additional 48 h in a low-attachment six-well plate to form tumorospheres (see Materials and Methods). Total protein lysates were prepared for western blot analysis, as indicated, including phospho-AKT (Ser473), phosphor-STAT3 (Ser727 and Tyr705), and phospho-NF-κB (Ser536) antibodies. CHC and β-actin antibodies were used for equal loading and transfer. Anti-FLAG antibodies were used for FLAG-tagged BRSK2 protein expression. n = 3, representative blots are shown. Duplicate BT-474 (A) and MDA-MB-231 (B) cell cultures were used to isolate nuclear and cytosolic fractions, free of nuclei, using established protocols. (E and F) As indicated, equal amounts of nuclear and cytosolic proteins were used for Western blotting, using specific antibodies against the target proteins. Translocation of p65-NF-κB or active STAT3(Y705) into the nucleus in response to ectopic BRSK2 or BRSK1 expression vs. empty vector was observed in both BT474 (E) and MDA-MB-231 (F) cell models. α-tubulin and total ERK1/2 antibodies, as well as histone H3 and Lamin B1 antibodies, were used to identify cytosolic and nuclear protein markers, respectively, free of nuclei. n = 3, representative blots were shown. Scanned blots and fold intensities normalized to respective loading control vs. vector control were labeled (ImageJ).
