Fig. 5. STK11 silencing attenuates HNK-mediated cytoprotective autophagy in breast cancer cells.

a Total protein lysates of MCF7 and MDA-MB-231 cells transfected with STK11^shRNA and control vector-pLKO.1 (vector) were immunoblotted for the expression of STK11. MCF7-pLKO.1 (vector) and STK11^shRNA cells were treated with 5 µM HNK for indicated times and immunoblotted for the expression of LC3B. b, c MCF7-pLKO.1 (vector), MCF7-STK11^shRNA, MDA-MB-231-pLKO.1 (vector) and MDA-MB-231-STK11^shRNA were treated with 5 µM HNK for 24 h and subjected to immunocytochemistry using LC3B antibody. Scale bars: 20 µm. Representative immunofluorescence images are shown. Bar graphs show number of LC3B puncta per cell. *P < 0.05. d, f MCF7-pLKO.1 control (vector) and STK11^shRNA cells were co-transfected with GFP-LC3B and RFP-RAB7 followed by treatment with 5 µM HNK for 24 h. Fixed cells were subjected to immunofluoroscent microscopy. Representative fluorescent images are shown. Scale bars: 10 µM. Bar graph shows number of red, green and yellow puncta per cell. *P < 0.05. e, g MCF7-pLKO.1 (vector) and STK11^shRNA cells were incubated with 10 µg/ml DQ-BSA for 2 h followed by treatment with 5 µM HNK for 24 h. Cells were fixed and stained with LysoTracker-Red followed by immunofluoroscent imaging. Representative images are shown. Scale bars: 10 µM. Bar graph shows number of yellow puncta per cell. *P < 0.05.