Cells expressing LC3-GFP-RFP were seeded on coverslips and incubated for 24 h at 37 °C. cells were treated with 1 µM Torin-1 (Selleckchem), 10 nM Bafilomycin A1 (Sigma-Aldrich), 10 µM JQ-1 (Medchem Express), 10 µM DT-061 (Medchem Express) or vehicle for 24 h, fixed with 4% PFA (Sigma-Aldrich). Nuclei were stained with DAPI. Coverslips were then mounted, and images were taken using a Leica SP8 AOBS (Leica microsystems) confocal microscopy using 63X oil objective and acquired with Leica Confocal Software. Quantification of dots number and colocalization were measured adapting the previously reported Red and Green Puncta Colocalization plugin and macro of ImageJ software [61 (link)].
Cells transfected with plasmids or siRNA were plated on glass coverslips were fixed with ice-cold methanol/acetone (1:1) for 2 min at −20 °C, washed and blocked with 5% BSA for 30 min. Cells were then incubated with primary antibodies for 45 min at room temperature. The following antibodies (specified in the Supplementary Information section) were used: anti-LC3 1:200, anti-LAMP2 1:100. Next, cells were incubated with Alexa Fluor (Life technologies) 488 or 647 fluorescent dye-conjugated secondary antibodies for 30 min at room temperature. Nuclei were counterstained with DAPI, mounted and analyzed by confocal microscopy Leica SP8 AOBS (Leica microsystems).
Cells transfected with plasmids or siRNA were plated on glass coverslips were fixed with ice-cold methanol/acetone (1:1) for 2 min at −20 °C, washed and blocked with 5% BSA for 30 min. Cells were then incubated with primary antibodies for 45 min at room temperature. The following antibodies (specified in the Supplementary Information section) were used: anti-LC3 1:200, anti-LAMP2 1:100. Next, cells were incubated with Alexa Fluor (Life technologies) 488 or 647 fluorescent dye-conjugated secondary antibodies for 30 min at room temperature. Nuclei were counterstained with DAPI, mounted and analyzed by confocal microscopy Leica SP8 AOBS (Leica microsystems).
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