Fig. 3: RNAseK knockout does not affect general lysosomal acidification or proteolytic activities. | Nature Communications

Fig. 3: RNAseK knockout does not affect general lysosomal acidification or proteolytic activities.

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

Fig. 3

a Quantification of LysoSensor Green signal fold change in control (sgControl) or RNAseK knockout (sgRNAseK) cells relative to signal in Control cells. MEF cells were left untreated or AA starved for 2 h and incubated with LysoSensor Green for 30 min followed by FACS analyses of fluorescence intensity. Mean + SEM is assessed by paired two-tailed Student’s t test. N = 3 biologically independent experiments. b Ratiometric pH quantification using Oregon Green 488-dextran. Control or RNAseK knockout cells were incubated with Oregon Green 488-dextran for 24 h, followed by assessment of fluorescence emission at 520 nm upon excitation at either 440 nm (pH insensitive) or 484 nm (pH sensitive). Calibration using standard buffers was used to determine lysosomal pH. Baf A1 was included as indicated. N = 30 cells examined over three independent experiments. c Western blot analyses of Cathepsin B in sgControl or sgRNAseK cells. Cells were untreated or AA starved in the presence or absence of Baf A1. d Quantification of mature and pro-Cathepsin B relative to sgControl cells in (c). N = 3 biologically independent experiments. e Schematic diagram of Cathepsin B activity assay where cleavage of Cathepsin B substrate results in the fluorescence of the cleaved product. e 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. f Quantification of Cathepsin B substrate fluorescence in the indicated cells. Signal was normalised relative to sgControl cells. MEF cells were left untreated or AA starved for 2 h and incubated with the Cathepsin B substrate for 30 min followed by FACS analyses. Mean + SEM is assessed by paired two-tailed Student’s t test. N = 3 biologically independent experiments. g Western blot analyses of EGFR levels in sgControl and sgRNAseK cells. Cells were cultured without serum for 4 h, followed by stimulation with EGF (20 ng/mL) for the indicated times. N = 3 biologically independent experiments. h Quantification of EGFR levels following EGF stimulation in (g) expressed as a percentage of the EGFR levels at time 0 in the relative cell line. N = 3 biologically independent experiments. In all panels, mean + SEM is assessed by unpaired two-tailed Student’s t-test, unless stated otherwise. Source data are provided with this paper.

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