Figure 3.
Arl8b depletion enhances recycling of the newly synthesized LAMP1 to the plasma membrane, impairing its lysosomal delivery. (A) Representative confocal micrographs of HeLa cells treated with control or Arl8b siRNA, transfected with SBP-GFP-LAMP1, followed by labeling with LTR (magenta). Images are taken at indicated time points after biotin addition. Scale bars: 10 µm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (***P = 0.0006). The values are represented as the mean ± SEM. (C) Schematic representation of the recycling assay showing endocytosis of anti-GFP antibody–labeled SBP-GFP-LAMP1 after 30 min of biotin addition, followed by acid wash to remove non-internalized anti-GFP antibody from the cell surface. Following this, pitstop2 (30 µM) was added to block endocytosis, and cells were incubated for 30 min to allow recycling of internalized anti-GFP antibody–bound SBP-GFP-LAMP1 back to the cell surface. Cells were fixed, and the surface staining was performed using an Alexa Fluor 568–conjugated secondary antibody without permeabilization. The schematic is created using BioRender. (D) Recycling assay was performed in HeLa cells treated with control or Arl8b siRNA, and representative micrographs depicting staining of surface-recycled SBP-GFP-LAMP1 are shown. Scale bar: 10 µm. (E) Graph represents the relative ratio of surface-recycled signal intensity (measured by anti-GFP antibody) to the total intensity of SBP-GFP-LAMP1 (total GFP signal) (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0048). The values are represented as the mean ± SEM. Refer to the image caption for details. Panel A shows confocal microscopy images of control or Arl8b siRNA-treated HeLa cells expressing SBP-GFP-LAMP1 and labeled with LysoTracker at multiple post-biotin incubation time points. Panel B shows a scatter bar plot quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and LysoTracker in control or Arl8b siRNA-treated HeLa cells after biotin addition. Panel C shows a schematic illustration of the recycling assay demonstrating endocytosis, acid wash removal, recycling, and surface detection of anti-GFP antibody-labeled SBP-GFP-LAMP1. Panel D shows fluorescence microscopy images of recycled surface SBP-GFP-LAMP1 in control or Arl8b siRNA-treated HeLa cells following recycling assay and pitstop2 treatment. Panel E shows a scatter bar plot quantifying the relative ratio of recycled surface anti-GFP signal intensity to total SBP-GFP-LAMP1 fluorescence intensity in control or Arl8b siRNA-treated HeLa cells.

Arl8b depletion enhances recycling of the newly synthesized LAMP1 to the plasma membrane, impairing its lysosomal delivery. (A) Representative confocal micrographs of HeLa cells treated with control or Arl8b siRNA, transfected with SBP-GFP-LAMP1, followed by labeling with LTR (magenta). Images are taken at indicated time points after biotin addition. Scale bars: 10 µm (main); 2 μm (inset). (B) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR in HeLa cells treated with indicated siRNAs is shown from three independent experiments (n = 3). Colors in the SuperPlots indicate individual experiments, with each dot representing a single cell. The mean value for each experiment is indicated by a larger dot. Statistical significance was calculated using unpaired Student’s t test (***P = 0.0006). The values are represented as the mean ± SEM. (C) Schematic representation of the recycling assay showing endocytosis of anti-GFP antibody–labeled SBP-GFP-LAMP1 after 30 min of biotin addition, followed by acid wash to remove non-internalized anti-GFP antibody from the cell surface. Following this, pitstop2 (30 µM) was added to block endocytosis, and cells were incubated for 30 min to allow recycling of internalized anti-GFP antibody–bound SBP-GFP-LAMP1 back to the cell surface. Cells were fixed, and the surface staining was performed using an Alexa Fluor 568–conjugated secondary antibody without permeabilization. The schematic is created using BioRender. (D) Recycling assay was performed in HeLa cells treated with control or Arl8b siRNA, and representative micrographs depicting staining of surface-recycled SBP-GFP-LAMP1 are shown. Scale bar: 10 µm. (E) Graph represents the relative ratio of surface-recycled signal intensity (measured by anti-GFP antibody) to the total intensity of SBP-GFP-LAMP1 (total GFP signal) (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0048). The values are represented as the mean ± SEM.

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