Mrfp lc3

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 method^{52 (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.

mRFP-LC3 (21075) and LAMP1-mGFP (34831) were obtained from Addgene. EGFP-LC3 and ATG7-siRNA plasmids were constructed by GeneChem Co. LTD (Shanghai, China). Plasmids were transfected into MDA-MB-231 and MCF-7 cells using Lipofectamine 3000 transfection reagent (Invitrogen, L3000). The transfection mixture was removed and replaced with fresh complete medium after 24 h incubation. The sequences of UBC9 shRNA were 5′-TTGGCAGTAAATCGTGTAGGCC-3′ (sh-UBC9–1#) and 5′-ATTTAGAAGTTCCTGTATTCCT-3′ (sh-UBC9–3#).
293FT cells were transfected with pLP1, pLP2, pLP/VSVG (Invitrogen, K4975) and sh-UBC9 or sh-Con plasmid. Supernatants containing the lentivirus were harvested after 48 h incubation and used to infect target cells. MDA-MB-231 and MCF-7 cells were selected with puromycin (4–8 μg/mL) (Sigma, P9620) to establish stable cell lines.