Increase in surface LAMP1 levels upon Arl8b depletion is independent of active lysosome exocytosis. (A) Lysates of HeLa, HEK293T, RPE-1, and THP-1 cells treated with control or Arl8b siRNA were IB with the indicated antibodies. The values indicate densitometric analysis of Arl8b levels normalized to α-tubulin. The bar graph represents the relative fold change in densitometric values of Arl8b normalized to α-tubulin. Statistical significance was calculated using the one-sample t test (n = 3; ***P = 0.0005 (HeLa); ***P = 0.0002 (THP-1); ****P < 0.0001). The values are represented as the mean ± SEM. (B) Wild-type or Arl8b KO HeLa cell lysates were IB for the indicated proteins. The values represent densitometric analysis of Arl8b levels normalized to α-tubulin. (C) Graph represents the MFI of surface LAMP2 and surface EGFR levels upon Arl8b siRNA treatment and normalized to control siRNA (n = 3). Statistical significance was calculated using the one-sample t test (n.s., non-significant). The values are represented as the mean ± SEM. (D) Representative histogram showing MFI of surface LAMP1 and LAMP2 in control or Arl8b siRNA-treated RPE-1, THP-1, and HEK293T cells as analyzed by flow cytometry. (E and F) Graphs represent the MFI of surface LAMP1 (E) (n = 3; each dot represents a single experiment, *P = 0.0488; n.s., non-significant) and LAMP2 (F) (n = 3; each dot represents a single experiment, *P = 0.0392; n.s., non-significant) upon Arl8b siRNA treatment and normalized to control siRNA. Statistical significance was calculated using the one-sample t test. The values are represented as the mean ± SEM. (G) Percentage of β-hexosaminidase release was quantified from the culture supernatant of HeLa cells treated with control or Arl8b siRNA upon ionomycin addition (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0092, n.s., non-significant). The values are represented as the mean ± SEM. (H) Cathepsin D levels in lysates and culture supernatants of HeLa cells treated with control or Arl8b siRNA upon ionomycin addition were measured by immunoblotting, and amido black staining was performed to visualize proteins. The values represent densitometric analysis of cathepsin D levels normalized to α-tubulin (lysate) or total protein (supernatant). (I) Representative confocal micrographs from live-cell imaging of HeLa cells expressing SBP-GFP-LAMP1 and lysosomes labeled with the SiR-Lysosome probe (magenta). Representative inset images at 30 and 90 min after biotin addition are shown. The line profiles indicate fluorescence intensity of SBP-GFP-LAMP1 (green) and SiR-Lysosome probe (magenta) along the yellow line. Scale bar: 2 µm. (J) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with the SiR-Lysosome probe at different time points 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 = 0.0012). The values are represented as the mean ± SEM. (K) Confocal micrographs from live-cell imaging of HeLa cells co-expressing SBP-GFP-LAMP1 and CD63-RFP at 30 and 90 min after biotin addition are shown. The line profiles indicate fluorescence intensity of SBP-GFP-LAMP1 (green) and CD63-RFP (magenta) along the yellow line. Scale bar: 2 µm. (L) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with CD63-RFP at different time points 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 = 0.0182). The values are represented as the mean ± SEM. (M) Confocal micrographs of HeLa cells expressing SBP-GFP-LAMP1 and immunostained for LAMP2 (magenta) at 30 and 90 min after biotin addition are shown. The line profiles indicate fluorescence intensity of SBP-GFP-LAMP1 (green) and LAMP2 (magenta) along the yellow line. Scale bar: 2 µm. (N) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with LAMP2 at different time points 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 = 0.0003). The values are represented as the mean ± SEM. (O) HeLa cells expressing SBP-GFP-LAMP1 (Y404A) are labeled with LTR (magenta). Confocal micrographs of live-cell imaging are shown at different time points after biotin addition. Scale bar: 10 µm. (P) Graph indicates Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 (Y404A) with the LTR 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 (n.s., non-significant). The values are represented as the mean ± SEM. Source data are available for this figure: SourceData FS1.