Figure 6.
RUFY1 regulates retrieval of CI-M6PR from endosomes to the TGN. (A–D) Representative confocal images of HeLa cells treated with the indicated siRNAs, followed by immunostaining for CI-M6PR and co-stained with Giantin or SNX1 (as labeled). Bars: (main) 10 µm; (insets) 2 µm. (E) Radial profile plot of the CI-M6PR intensity distribution in HeLa cells treated with indicated siRNAs. The values plotted are the mean ± SD from three independent experiments, with 30–40 cells analyzed per experiment. (F and G) Pearson’s and Mander’s colocalization coefficient quantification of CI-M6PR with Giantin and SNX1 (as indicated in the graph) in control siRNA and RUFY1 siRNA-treated HeLa cells. The values plotted are the mean ± SD from three independent experiments. Experiments are color coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; unpaired two-tailed t test). (H and I) Representative confocal micrographs of HeLa cells treated with the indicated siRNA and transfected with the RUFY1-GFP (Rescue) construct, followed by immunostaining with an anti-CI-M6PR antibody. Cells expressing rescue construct are marked with a white boundary. Bars: 10 µm. (J) Radial profile plot of the CI-M6PR intensity distribution in HeLa cells treated with the indicated siRNAs and transfected with the RUFY1-GFP (Rescue) construct. The values plotted are the mean ± SD from three independent experiments, with 30–40 cells analyzed per experiment. (K) Schematic illustrating CD8α-M6PR trafficking assay. The anti-CD8 primary antibody was used for labeling the cell surface receptor population, followed by a chase of the internalized CD8α-M6PR for either 5 min or 60 min. Cells were fixed and immunostained with secondary antibodies. (L) Pearson’s colocalization coefficient quantification of endocytosed CD8α-M6PR with EEA1 and Giantin markers after 5 min of chase. Cells were transfected with CD8α-M6PR chimera construct after 60 h of the indicated siRNA treatments, followed by labeling cell surface CD8α-M6PR receptors and chase for 5 min post internalization. Cells were fixed and immunostained for EEA1 and Giantin. (M–P) Representative confocal images of endocytosed CD8α-M6PR at 60 min post internalization. Cells were fixed and immunostained for EEA1 and Giantin. Bars: (main) 10 µm; (insets) 2 µm. (Q) Pearson’s colocalization coefficient quantification of endocytosed CD8α-M6PR with EEA1 and Giantin markers at 60 min of the chase in HeLa cells treated with indicated siRNA. For L and Q, the values plotted are the mean ± SD from three independent experiments. Experiments are color coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; n.s., not significant; unpaired two-tailed t test).

RUFY1 regulates retrieval of CI-M6PR from endosomes to the TGN. (A–D) Representative confocal images of HeLa cells treated with the indicated siRNAs, followed by immunostaining for CI-M6PR and co-stained with Giantin or SNX1 (as labeled). Bars: (main) 10 µm; (insets) 2 µm. (E) Radial profile plot of the CI-M6PR intensity distribution in HeLa cells treated with indicated siRNAs. The values plotted are the mean ± SD from three independent experiments, with 30–40 cells analyzed per experiment. (F and G) Pearson’s and Mander’s colocalization coefficient quantification of CI-M6PR with Giantin and SNX1 (as indicated in the graph) in control siRNA and RUFY1 siRNA-treated HeLa cells. The values plotted are the mean ± SD from three independent experiments. Experiments are color coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; unpaired two-tailed t test). (H and I) Representative confocal micrographs of HeLa cells treated with the indicated siRNA and transfected with the RUFY1-GFP (Rescue) construct, followed by immunostaining with an anti-CI-M6PR antibody. Cells expressing rescue construct are marked with a white boundary. Bars: 10 µm. (J) Radial profile plot of the CI-M6PR intensity distribution in HeLa cells treated with the indicated siRNAs and transfected with the RUFY1-GFP (Rescue) construct. The values plotted are the mean ± SD from three independent experiments, with 30–40 cells analyzed per experiment. (K) Schematic illustrating CD8α-M6PR trafficking assay. The anti-CD8 primary antibody was used for labeling the cell surface receptor population, followed by a chase of the internalized CD8α-M6PR for either 5 min or 60 min. Cells were fixed and immunostained with secondary antibodies. (L) Pearson’s colocalization coefficient quantification of endocytosed CD8α-M6PR with EEA1 and Giantin markers after 5 min of chase. Cells were transfected with CD8α-M6PR chimera construct after 60 h of the indicated siRNA treatments, followed by labeling cell surface CD8α-M6PR receptors and chase for 5 min post internalization. Cells were fixed and immunostained for EEA1 and Giantin. (M–P) Representative confocal images of endocytosed CD8α-M6PR at 60 min post internalization. Cells were fixed and immunostained for EEA1 and Giantin. Bars: (main) 10 µm; (insets) 2 µm. (Q) Pearson’s colocalization coefficient quantification of endocytosed CD8α-M6PR with EEA1 and Giantin markers at 60 min of the chase in HeLa cells treated with indicated siRNA. For L and Q, the values plotted are the mean ± SD from three independent experiments. Experiments are color coded, and each dot represents the individual data points from each experiment. The total number of cells analyzed is indicated on the top of each data set (****P < 0.0001; n.s., not significant; unpaired two-tailed t test).

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