Fig. 2: RNAseK knockout results in the accumulation of autolysosomal structures. | Nature Communications

Fig. 2: RNAseK knockout results in the accumulation of autolysosomal structures.

From: The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Fig. 2

a Representative immunofluorescence image of GFP-LC3 and endogenous LAMP1 in sgControl or sgRNAseK MEFs cultured in the absence of AA for 2 h. Scale bar: 10 µm. Quantification of PCC between GFP-LC3 and LAMP1 is shown on the right. N = 30 cells from three independent experiments. b Representative immunofluorescence image of endogenous p62 and LAMP1 in siControl or siRNAseK MEFs treated as in (a). Scale bar: 10 µm. Quantification of PCC between p62 and LAMP1 is shown on the right. N = 30 cells from three independent experiments. c Representative electron microscopy images of sgControl and sgRNAseK U2OS cells treated in the absence of AA for 3 h. Baf A1 is added as indicated. Arrows indicate autophagosomes (white arrows) and autolysosomes (black arrows). Scale bar: 1 µm. N = 1. d Model of studying IAM degradation whereby STX17 association with undegraded or degraded autolysosomes are observed as a lysotracker ring or dots, respectively. d was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. e Representative images of sgControl and sgRNAseK MEFs stably expressing GFP-STX17 and stained with Lysotracker red. Quantifications of the percentage of STX17 with Lysotracker rings or dots are shown on the right. Scale bar: 10 µm. N = 15 cells from three independent experiments. In all panels, mean + SEM is assessed by unpaired two-tailed Student’s t test. Source data are provided with this paper.

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