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. 2020 Dec 7;117(51):32443–32452. doi: 10.1073/pnas.2011442117

Fig. 4.

Fig. 4.

Knockdown of macroautophagy/CMA machinery does not affect lipid/protein transfer between lysosomes and LDs. AML12 hepatocytes were treated for 72 h with a nontargeting control siRNA (siNT), siAtg5, siLAMP2A, or a combination of both siAtg5 and siLAMP2A, to examine possible compensation between these pathways. Cells were treated for 24 h ± 50 μM lalistat and cultured for 4 h in full-serum (10% FBS) or HBSS starvation conditions. (A) Confocal live-cell imaging stills of siNT-treated cells cultured under full-serum conditions ±50 μM lalistat. Lysosomes are stained in magenta with LysoTracker Deep Red, and LDs are stained with LipidTOX green. Note the accumulation of lipid within lysosomes upon 24 h LAL inhibitor (LALi) treatment (arrows). (B) Quantification of lysosomal lipid accumulation from live cells. LALi treatment results in significant retention of lipids within the lysosome. Knockdowns of macroautophagy or CMA components do not perturb this lipid accumulation. Summary of n = 3 independent experiments in which >5 fields of ∼40 cells were quantified. *P < 0.05 as measured by paired t test. Error bars reflect SD. NS, not significant. (C and D) siRNA-mediated knockdown of ATGL negatively affects lysosomal lipid accumulation. Confocal images (C) using the same uptake assay conditions as in A, reveals a ∼40% reduction (D) in lysosomal lipid uptake upon ATGL knockdown. Summary of n = 3 independent experiments in which >5 fields of ∼40 cells were quantified. *P < 0.05, **P < 0.01 as measured by paired t test. Error bars reflect SD. (E) Immunoblotting to demonstrate the knockdown efficiency of siATGL treatment in the experiments used in C and D. (F) Confocal images of AML12 mouse hepatocytes transiently expressing the PLIN2-mRFP1-EGFP reporter and subjected to 72-h treatment with a nontargeting control siRNA (siNT), siAtg5, or siLAMP2A shows the presence of RFP+-only puncta in all three cases. Insets show individual RFP+ and EGFP channels with arrows indicating RFP+-only puncta. (G) Quantification of the number of RFP+-only puncta from >10 cells from n = 3 independent experiments revealed no significant difference in the ability of the reporter to be internalized into the lysosome when macroautophagy or CMA machinery was compromised by 72-h treatment with indicated siRNAs. NS, not significant. (H) Immunoblotting to demonstrate the knockdown efficiency of siAtg5 or siLAMP2A treatment in the experiments used in F and G.