Mitochondrial or ER staining was achieved by transient transfection of plasmids pmTurquoise2-Mito (Addgene plasmid #36208) or pmTurquoise2-ER (Addgene plasmid #36204) (gift from Dorus Gadella)17 (link). These were co-transfected with plasmid mRFP-LC3 (Addgene plasmid #21074; kindly provided by Pr. T. Yoshimori)13 (link).
TFEB imaging was achieved by transient transfection of plasmids pEGFP-N1-TFEB (gift from Shawn Ferguson) (Addgene plasmid # 38119)51 (link). Quantitative assessment of the TFEB nuclear localization was achieved by dividing the density of the nuclear signal by the mean density of the fluorescent signal in each cell using the ImageJ software. Fifteen images per condition from five distinct experiments were assessed.
Ad-LC3 imaging was achieved 48 h post infection (MOI1) on INS-1E cells plated on glass cell culture chamber (Ibidi) and photographed on a Leica inverted microscope (×40 oil immersion fluorescence objective). The Ad-LC3 green and red stainings were first converted to a 16 bit format and the signal to noise ratio was determined by applying the Yen threshold method52 (link). A binary image was then created and the number of particles (LC3 dots) was measured. Data were normalized to the number of cells in each image.
TFEB imaging was achieved by transient transfection of plasmids pEGFP-N1-TFEB (gift from Shawn Ferguson) (Addgene plasmid # 38119)51 (link). Quantitative assessment of the TFEB nuclear localization was achieved by dividing the density of the nuclear signal by the mean density of the fluorescent signal in each cell using the ImageJ software. Fifteen images per condition from five distinct experiments were assessed.
Ad-LC3 imaging was achieved 48 h post infection (MOI1) on INS-1E cells plated on glass cell culture chamber (Ibidi) and photographed on a Leica inverted microscope (×40 oil immersion fluorescence objective). The Ad-LC3 green and red stainings were first converted to a 16 bit format and the signal to noise ratio was determined by applying the Yen threshold method52 (link). A binary image was then created and the number of particles (LC3 dots) was measured. Data were normalized to the number of cells in each image.
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