Figure 7.
RUFY1 depletion impairs CI-M6PR cargo, cathepsin Z, delivery to lysosomes. (A) Schematic representation of the RUSH assay to study the trafficking of CI-M6PR cargo, mCherry-Cathepsin Z (Cath Z), from the endoplasmic reticulum (ER) to the lysosome. SBP-mCherry-CathZ is retained in the ER via its interaction with the hook Str-KDEL. Upon biotin addition, mCherry-Cath Z releases from the ER and over time reaches the Golgi and subsequently to the lysosomes. (B–I) Representative confocal images of a RUSH experiment performed in HeLa cells treated with control or RUFY1 siRNA. Following 60 h of indicated siRNA treatment; cells were co-transfected with Str-KDEL-IRES-SBP-mCherry-Cath Z and LAMP1-GFP constructs. Different transfected control and RUFY1 depleted cells were imaged at 37°C prior to biotin addition and at various time points post biotin addition. Representative images of different cells at 40, 180, and 320 min post biotin addition are shown. Bars: (main) 10 µm; (insets) 2 µm. (J) Pearson’s colocalization coefficient was quantified for the cargo, mCherry-Cath Z, with LAMP1-GFP at different time points after the addition of biotin by drawing different ROIs in cells. Data represents mean ± SD from 30 to 40 cells in total from three independent experiments, and in each cell, 2–3 ROI were selected for analysis (****P < 0.0001; **P < 0.01; *P < 0.05; unpaired two-tailed t test). (K) Immunoblot of pro-cathepsin D secretion assay performed in HeLa cells treated with the indicated siRNA. TCA precipitated proteins from cell culture media of siRNA-treated cells were immunoblotted with an anti-cathepsin D antibody. Ponceau S stain was done to visualize equal loading of proteins. (L) Densitometric analysis of pro-cathepsin D band intensity in culture media normalized to mature cathepsin and α-tubulin band intensity in total cell lysates. The averaged values from two independent experiments are plotted. Source data are available for this figure: SourceData F7.

RUFY1 depletion impairs CI-M6PR cargo, cathepsin Z, delivery to lysosomes. (A) Schematic representation of the RUSH assay to study the trafficking of CI-M6PR cargo, mCherry-Cathepsin Z (Cath Z), from the endoplasmic reticulum (ER) to the lysosome. SBP-mCherry-CathZ is retained in the ER via its interaction with the hook Str-KDEL. Upon biotin addition, mCherry-Cath Z releases from the ER and over time reaches the Golgi and subsequently to the lysosomes. (B–I) Representative confocal images of a RUSH experiment performed in HeLa cells treated with control or RUFY1 siRNA. Following 60 h of indicated siRNA treatment; cells were co-transfected with Str-KDEL-IRES-SBP-mCherry-Cath Z and LAMP1-GFP constructs. Different transfected control and RUFY1 depleted cells were imaged at 37°C prior to biotin addition and at various time points post biotin addition. Representative images of different cells at 40, 180, and 320 min post biotin addition are shown. Bars: (main) 10 µm; (insets) 2 µm. (J) Pearson’s colocalization coefficient was quantified for the cargo, mCherry-Cath Z, with LAMP1-GFP at different time points after the addition of biotin by drawing different ROIs in cells. Data represents mean ± SD from 30 to 40 cells in total from three independent experiments, and in each cell, 2–3 ROI were selected for analysis (****P < 0.0001; **P < 0.01; *P < 0.05; unpaired two-tailed t test). (K) Immunoblot of pro-cathepsin D secretion assay performed in HeLa cells treated with the indicated siRNA. TCA precipitated proteins from cell culture media of siRNA-treated cells were immunoblotted with an anti-cathepsin D antibody. Ponceau S stain was done to visualize equal loading of proteins. (L) Densitometric analysis of pro-cathepsin D band intensity in culture media normalized to mature cathepsin and α-tubulin band intensity in total cell lysates. The averaged values from two independent experiments are plotted. Source data are available for this figure: SourceData F7.

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