Journal: Biomolecules

Article Title: Isolation, Characterization, and Autophagy Function of BECN1-Splicing Isoforms in Cancer Cells

doi: 10.3390/biom12081069

Figure Lengend Snippet: BECN1- α interacts with PRKN stimulating mitophagy. ( A ) MDA-MB231 cells plated on coverslips were transfected with pEGFP-N1 vector carrying BECN1-wt-GFP (wt-GFP), BECN1-α-GFP (α-GFP), BECN1-β-GFP (β-GFP), or BECN1-γ-GFP (γ-GFP). After 48 h, cells were fixed, stained for PRKN (red) and imaged by florescence microscopy. Where indicated, cells were treated with 10 μM CCCP for the last 3 h. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. Histograms show the intensity densities of yellow signals (Int DEN), which result from close green and red fluorescence and are representative of the level of colocalization between PRKN and GFP. Asterisks indicate significantly different yellow signal intensity (**** p < 0.0001). Error bars, SD. ( B ) MDA-MB231 were plated on coverslips and transfected with pEGFP-N1 vector carrying BECN1-wt-GFP (wt-GFP) or BECN1-α-GFP (α-GFP). After 48 h, cells were fixed, stained for LC3 (red) and BNIP3 (green), and imaged by fluorescence microscopy. Where indicated, cells were treated with 10 μM CCCP for the last 3 h. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. Histograms show the intensity densities of yellow signals (Int DEN), which result from close green and red fluorescence and are representative of the level of colocalization between BNIP3 and LC3. Asterisks indicate significantly different yellow signal intensity (* p < 0.05). Error bars, SD. ( C ) MDA-MB231 were plated on coverslips and transfected and treated as in (B). After 48 h, cells were incubated with 500 nM Mitotracker™ RED (MitoRed) for the last 15 min at 37 °C. Following Mitotracker incubation, cells were washed with PBS, fixed, stained for LC3 (cyan), and imaged by fluorescence microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. Histograms show the intensity densities of white signals (Int DEN), which result from close red and cyan fluorescence and are representative of the level of colocalization between Mitotracker and LC3. Asterisks indicate significantly different white signal intensity (* p < 0.05). Error bars, SD. ( D ) MDA-MB231 cells were plated on Petri dishes and transfected with pEGFP-N1 vector carrying BECN1-α-GFP (α-GFP). After 48 h, cells were harvested and processed for immunoblotting with the indicated antibodies. Where indicated, cells were treated with 10 μM CCCP for the last 3 h. Band intensities were determined by densitometric analysis and GFP/Actin, AMBRA1/Actin, PINK1/Actin, PRKN/Actin, BNIP3/Actin ratios are shown. ( E ) Cell homogenates from ( C ) were processed for the immunoprecipitation of BECN1-α-GFP isoform, as described in the Materials and Methods section. Immune complexes were separated by SDS-PAGE and processed for immunoblotting with the indicated antibodies.

Article Snippet: The following primary antibodies were employed in the immunofluorescence or immunoblotting studies: rabbit polyclonal anti-BECN1 (PA5-96649, Invitrogen), mouse monoclonal anti-Bcl2 (15071S, Cell-Signaling, Danvers, MA, USA), rabbit monoclonal anti-Vps34 (4263S, Cell-Signaling), rabbit polyclonal anti-ATG14 (SAB1306130, Sigma-Aldrich), rabbit monoclonal anti-UVRAG (5320S, Cell-Signaling), rabbit polyclonal anti-AMBRA1 (PA5-88053, Invitrogen), rabbit polyclonal anti-PINK1 (NB100-493, Novus, St. Louis, MO, USA), rabbit polyclonal anti-PRKN (NB100-91921, Novus), rabbit monoclonal anti-BNIP3 (44060S, Cell-Signaling), mouse monoclonal anti-GFP (632381, Clontech), mouse monoclonal anti-GFP (2955S, Cell-Signaling), rabbit polyclonal anti-LC3 (L7543, Sigma-Aldrich), mouse monoclonal anti-Lamp1 (555798, BD Bioscience, Franklin Lakes, NJ, USA), mouse monoclonal anti-p62 (MABC32, Millipore, Burlington, MA, USA), mouse monoclonal anti-β-Actin (A5441, Sigma-Aldrich), mouse monoclonal anti- β-Tubulin (T5201, Sigma-Aldrich).

Techniques: Transfection, Plasmid Preparation, Staining, Microscopy, Fluorescence, Incubation, Western Blot, Immunoprecipitation, SDS Page