Fig. 5: PLD3 is required for autophagy. | Nature Communications

Fig. 5: PLD3 is required for autophagy.

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

Fig. 5

a Quantitative proteomic analysis of enriched lysosomal fractions derived from sgControl or sgRNAseK MEFs. The statistical significance was calculated by unpaired two-tailed Student’s t test. b MS analyses of PLD3 levels in cell media derived from sgControl or sgRNAseK MEFs. Baf A1 treatment was added as indicated (24 h). Absolute PLD3 intensity is shown as label-free quantitation (LFQ) values. N = 1 including three technical repeats. c qRT-PCR analyses of mRNA from MEFs transfected with non-targetting siRNA (siControl) or siPLD3. Pld3 mRNA levels were normalised to Actin. Fold change in expression was compared to siControl cells. N = 3 biologically independent experiments. d Western blot analyses of cell lysates derived from siControl MEFs or cells transfected with siRNA sequences targeting PLD3. Baf A1 treatment (2 h) was included as indicated. Quantification of p62 and LC3-II band intensity normalised relative to siControl (lane 1) is shown. N = 3. e Western blot analyses of lysates derived from SH-SY5Y cells transfected with siControl or pool siRNAs targeting RNAseK or PLD3. Baf A1 treatment (3 h) was included as indicated. Quantification of LC3-II band intensity normalised relative to siControl (lane 1) is shown. N = 3. f Representative electron microscopy images of siControl and siPLD3 MEFs treated in the absence of AA (3 h). Arrows indicate autophagosomes (white) and autolysosomes (black). Scale bar: 1 µm. N = 1. g Representative immunofluorescence images of MEFs transfected with siControl or siPLD3. Cells were cultured in the absence of AA (2 h). Scale bar: 10 µm. Quantification of PCC between p62 and LAMP1 in siControl and siPLD3 is shown on the right. N = 30 cells from three independent experiments. h Representative images of sgControl and sgPLD3 MEFs 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. i Western blot analyses of EGFR levels in siControl and siPLD3 MEF cells. Cells were cultured without serum (4 h), followed by stimulation with EGF (20 ng/mL). Quantification of EGFR levels as a percentage of time 0 is shown on the right. N = 3 biologically 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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