Publication protocol
Cellular localization of autophagy biomarkers and NFE2L2
Subcellular localization of SQSTM1, NFE2L2 and LC3B was determined by immunostaining and visualization by fluorescently labeled secondary antibodies. Immunostaining was imaged by an Axiovert200 microscope equipped with a 63×1.2W objective and the confocal module LSM510 META (Carl Zeiss). Images were processed using the LSM software (Carl Zeiss) and mounted using Canvas 11 (ACD Systems). Images are representative of more than 200 randomly selected cells in each condition and of two or more independent experiments. Cytoplasmic structures enriched for LC3 were quantified by using the histogram function in the LSM software. All images were taken with the same settings. Pixels positive for LC3B with an intensity of more than 100 RLU were counted and divided by the number of cells in the image (more than 100 cells per condition).
Autophagy determined by flow cytometry
Autophagic structures were determined using the CytoID autophagy detection kit following the suppliers’ instructions (Enzo Life Sciences). Briefly, the cells were detached by trypsination and stained in a solution with Cyto-ID Detection Reagent covered from light for 30 min at room temperature with gentle rotation, and the fluorescent intensity per cell was determined using a flow cytometer.
Immunoblotting
Cells, transfected or non-transfected, untreated or treated with DHA and/or 100 nM BafA1, were harvested by trypsinization or scraped in ice cold PBS. Cells were lysed in either a buffer containing 1% NP40, 0.25% Triton-X100, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA pH 8, Complete® protease inhibitor (PI, Roche), and phosphatase inhibitor cocktail I and III (Sigma-Aldrich), or an urea buffer containing 8 M Urea, 0.5% (v/v) Triton X-100, 100 mM DTT, PI, and phosphatase inhibitor cocktail I and III. Total protein concentration in the lysates was determined by Bio-Rad protein assay (Bio-Rad). Bound antibodies were imaged by near infrared fluorescence using appropriate fluorescent dye labeled secondary antibodies and an Odyssey NIR scanner (Li-Cor Biosciences). Images were processed using the Li-Cor Odyssey software and mounted using the Canvas 11 software (ACD Systems).
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