![Arl8b depletion leads to an increase in surface LAMP1 levels and characterization of newly synthesized LAMP1 trafficking to active lysosomes. (A) Representative histogram showing MFI of surface LAMP1 in control or Arl8b siRNA-treated HeLa cells as analyzed by flow cytometry. (B) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with indicated siRNAs normalized to control siRNA from three independent experiments (n = 3). Statistical significance was calculated using one-sample t test (*P = 0.0122). The values are represented as the mean ± SEM. (C) Representative histogram of wild-type (WT) or Arl8b KO HeLa cells showing MFI of surface LAMP1 and LAMP2 as analyzed by flow cytometry. (D) Bar graph represents the percent MFI of surface LAMP1 and LAMP2 in Arl8b KO cells normalized to WT cells from four independent experiments (n = 4). Statistical significance was calculated using one-sample t test (**P = 0.0064 [LAMP1]; **P = 0.0095 [LAMP2]). The values are represented as the mean ± SEM. (E) Schematic depiction of the RUSH assay used to investigate the trafficking of newly synthesized LAMP1. ER hook streptavidin-KDEL, along with a reporter (LAMP1) fused with SBP-GFP, is expressed in HeLa cells. The KDEL hook enables retention of the reporter in the ER through interaction between streptavidin and SBP. Biotin addition allows the reporter to be released and subsequently trafficked via the secretory pathway. The schematic is created using BioRender. (F) Representative confocal micrographs from a live-cell imaging experiment of HeLa cells expressing the RUSH reporter (SBP-GFP-LAMP1) and labeled with LTR. The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and LTR (magenta). Scale bars: 10 μm (main); 2 μm (inset). (G) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR 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 = 0.0022). The values are represented as the mean ± SEM. (H) Representative live-cell time-lapse images of HeLa cells expressing SBP-GFP LAMP1 (green) and LTR (magenta) after 80 min of biotin addition. The yellow arrowheads indicate SBP-GFP-LAMP1– and LTR-positive vesicles undergoing kiss-and-run events. Scale bar: 2 µm. (I) Representative confocal micrographs of HeLa cells expressing SBP-GFP-LAMP1. Cells were incubated with mCherry-tagged anti-GFP nanobody at the time of biotin addition, followed by fixation at the indicated time points. Scale bars: 10 μm (main); 2 μm (inset). (J) PCC of SBP-GFP-LAMP1 with mCherry-tagged anti-GFP nanobody at different time points of biotin addition 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 by one-way ANOVA with Dunnett’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. MFI, mean fluorescence intensity; PCC, pearson's correlation coefficient. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jcb/225/7/10.1083_jcb.202509040/1/jcb_202509040_fig1.png?Expires=1782456221&Signature=huU3Mfaw3pMHakwohBCd6QzRCgZ-ab539g5sPXYXF-F4Oldpb~km18PLlwDJsWQLY5s2aWonY0aSB9YGbuZDjWiy5xZPv3Q4nTYaQHPFzAjezWqVmI-LuhYhKBTQlf-~Ak7J45jYDtGWIqYGWbo7R0Kcj5ZcW5UMz560LXj3A5bZ6RZaLeBjDMXawNYQBvS9wPw6ovk-z-jAs8QvZGd52ekNWqSibw8G9y1N-v3fdUau5LT8OA2UpanD3D8QwceukfkuUh3fSk~PgE1BqRYXpCm5aY~VJcn8zb4yRM-e9sk7pHgv3grsL~iyeYfZZoZnnIZNHZqz0F4sS8sFYjSr9w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows a representative flow cytometry histogram comparing mean fluorescence intensity of surface LAMP1 in control and Arl8b siRNA-treated HeLa cells. Panel B shows a bar graph quantifying percentage mean fluorescence intensity of surface LAMP1 in HeLa cells treated with indicated siRNAs normalized to control siRNA. Panel C shows representative flow cytometry histograms comparing surface LAMP1 and surface LAMP2 intensity between wild-type and Arl8b knockout HeLa cells. Panel D shows bar graphs quantifying percentage mean fluorescence intensity of surface LAMP1 and surface LAMP2 in Arl8b knockout cells normalized to wild-type cells. Panel E shows a schematic illustration of the RUSH assay demonstrating trafficking of newly synthesized SBP-GFP-LAMP1 from the endoplasmic reticulum through the secretory pathway following biotin addition. Panel F shows live-cell confocal microscopy images and fluorescence intensity line profiles of SBP-GFP-LAMP1 colocalization with LysoTracker at different time points following biotin addition. Panel G shows scatter dot plots quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and LysoTracker from multiple independent live-cell imaging experiments. Panel H shows representative live-cell time-lapse microscopy images of SBP-GFP-LAMP1 and LysoTracker positive vesicles undergoing kiss-and-run interactions after biotin addition. Panel I shows confocal microscopy images of HeLa cells expressing SBP-GFP-LAMP1 incubated with mCherry-tagged anti-GFP nanobody and fixed at indicated post-biotin time points. Panel J shows scatter bar plots quantifying Pearsons correlation coefficient between SBP-GFP-LAMP1 and mCherry-tagged anti-GFP nanobody at different biotin incubation time points.
Arl8b depletion leads to an increase in surface LAMP1 levels and characterization of newly synthesized LAMP1 trafficking to active lysosomes. (A) Representative histogram showing MFI of surface LAMP1 in control or Arl8b siRNA-treated HeLa cells as analyzed by flow cytometry. (B) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with indicated siRNAs normalized to control siRNA from three independent experiments (n = 3). Statistical significance was calculated using one-sample t test (*P = 0.0122). The values are represented as the mean ± SEM. (C) Representative histogram of wild-type (WT) or Arl8b KO HeLa cells showing MFI of surface LAMP1 and LAMP2 as analyzed by flow cytometry. (D) Bar graph represents the percent MFI of surface LAMP1 and LAMP2 in Arl8b KO cells normalized to WT cells from four independent experiments (n = 4). Statistical significance was calculated using one-sample t test (**P = 0.0064 [LAMP1]; **P = 0.0095 [LAMP2]). The values are represented as the mean ± SEM. (E) Schematic depiction of the RUSH assay used to investigate the trafficking of newly synthesized LAMP1. ER hook streptavidin-KDEL, along with a reporter (LAMP1) fused with SBP-GFP, is expressed in HeLa cells. The KDEL hook enables retention of the reporter in the ER through interaction between streptavidin and SBP. Biotin addition allows the reporter to be released and subsequently trafficked via the secretory pathway. The schematic is created using BioRender. (F) Representative confocal micrographs from a live-cell imaging experiment of HeLa cells expressing the RUSH reporter (SBP-GFP-LAMP1) and labeled with LTR. The line profiles indicate fluorescence intensity along the yellow lines for both channels: SBP-GFP-LAMP1 (green) and LTR (magenta). Scale bars: 10 μm (main); 2 μm (inset). (G) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LTR 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 = 0.0022). The values are represented as the mean ± SEM. (H) Representative live-cell time-lapse images of HeLa cells expressing SBP-GFP LAMP1 (green) and LTR (magenta) after 80 min of biotin addition. The yellow arrowheads indicate SBP-GFP-LAMP1– and LTR-positive vesicles undergoing kiss-and-run events. Scale bar: 2 µm. (I) Representative confocal micrographs of HeLa cells expressing SBP-GFP-LAMP1. Cells were incubated with mCherry-tagged anti-GFP nanobody at the time of biotin addition, followed by fixation at the indicated time points. Scale bars: 10 μm (main); 2 μm (inset). (J) PCC of SBP-GFP-LAMP1 with mCherry-tagged anti-GFP nanobody at different time points of biotin addition 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 by one-way ANOVA with Dunnett’s multiple comparisons test (****P < 0.0001). The values are represented as the mean ± SEM. MFI, mean fluorescence intensity; PCC, pearson's correlation coefficient.