Figure S3.
Arl8b-positive vesicles transiently interact with AP-3–positive endosomes, which represent the sorting station for the newly synthesized LAMP1 to active lysosomes. (A) HeLa cell lysates treated with control or AP-3 siRNA were IB for the indicated proteins. The values represent densitometric analysis of AP-3 levels normalized to α-tubulin. (B) Representative confocal micrographs of HeLa cells treated with control or AP-3 siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Live-cell imaging was performed after cells were incubated with LTR (magenta) dye to label acidic lysosomes, followed by biotin addition. Yellow arrowheads in the insets denote the colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (C) Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 with LTR in HeLa cells treated with control or AP-3 siRNA 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 < 0.0001). The values are represented as the mean ± SEM. (D) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with the indicated siRNAs and normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0050). The values are represented as the mean ± SEM. (E) Representative confocal images of HeLa cells treated with control or AP-3 siRNA, followed by co-expression of SBP-GFP-LAMP1 (green) and Arl8b-Halo (magenta). Cells were incubated with biotin, and live-cell imaging was performed. Scale bars: 10 μm (main); 2 μm (inset). (F) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Arl8b-Halo in HeLa cells treated with the 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 (n.s., non-significant). The values are represented as the mean ± SEM. (G) Representative confocal micrographs of Arl8bEN-mStayGold KI (green) HeLa cells immunostained for endogenous AP-3 (magenta). Yellow arrowheads in the insets denote the colocalized pixels. The line profiles indicate fluorescence intensity along the yellow lines for both channels: Arl8bEN-mStayGold (green) and AP-3 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (H) Quantification of Manders’ colocalization coefficient (M1 and M2) of Arl8bEN-mStayGold with AP-3 in HeLa cells 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. The values are represented as the mean ± SEM. (I) Representative confocal micrograph from a live-cell imaging experiment of HeLa cells co-expressing Arl8b-GFP (green) and AP-3 (M1)-mScarlet (magenta); insets show time-lapse images. Micrographs are maximum-intensity projections of z-stack images from a live-cell video. Yellow arrowheads in the insets denote the colocalized pixels. The line profiles indicate fluorescence intensity along the yellow lines for both channels: Arl8b-GFP (green) and AP-3 (M1)-mScarlet (magenta). Scale bars: 10 μm (main); 2 μm (inset). (J) Representative confocal micrographs of HeLa cells immunostained for endogenous AP-3 (magenta) and Rab14 (green) (left panel). Representative confocal images of HeLa cells expressing RUFY1-FLAG were immunostained with anti-FLAG (green) and anti-AP-3 antibodies (magenta) (right panel). Yellow arrowheads in the insets denote the colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (K) Quantification of Manders’ colocalization coefficient of AP-3 with Rab14 and RUFY1-FLAG, respectively, 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. The values are represented as the mean ± SEM. (L) Representative confocal images of HeLa cells treated with control or Arl8b siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Cells were fixed at 60 min after biotin addition and immunostained for endogenous Rab14 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (M) HeLa cell lysates treated with the control or Rab11a siRNAs were IB for the indicated proteins. The values represent densitometric analysis of Rab11a levels normalized to α-tubulin. Source data are available for this figure: SourceData FS3. Refer to the image caption for details. Panel A shows immunoblot images of HeLa cell lysates treated with control or AP-3 siRNA, indicating protein levels of AP-3 and α-tubulin. Panel B shows confocal microscopy images of control or AP-3 siRNA-treated HeLa cells expressing SBP-GFP-LAMP1 and labeled with LysoTracker, highlighting colocalization regions. Panel C shows scatter bar plots quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and LysoTracker in control or AP-3 siRNA-treated cells. Panel D shows a scatter bar plot comparing relative mean fluorescence intensity of surface LAMP1 in control and AP-3 siRNA-treated HeLa cells. Panel E shows confocal microscopy images of control or AP-3 siRNA-treated HeLa cells co-expressing SBP-GFP-LAMP1 and Arl8b-Halo during live-cell imaging. Panel F shows scatter bar plots quantifying Manders colocalization coefficient between SBP-GFP-LAMP1 and Arl8b-Halo in control or AP-3 siRNA-treated cells. Panel G shows confocal microscopy images and fluorescence intensity line profiles of Arl8bEN-mStayGold knock-in HeLa cells immunostained for endogenous AP-3. Panel H shows scatter bar plots quantifying Manders colocalization coefficients between Arl8bEN-mStayGold and AP-3 using M1 and M2 measurements. Panel I shows live-cell time-lapse confocal microscopy images and fluorescence intensity line profiles of HeLa cells co-expressing Arl8b-GFP and AP-3-mScarlet. Panel J shows confocal microscopy images of HeLa cells stained for AP-3 with Rab14 or expressing RUFY1-FLAG with AP-3, highlighting colocalization regions. Panel K shows scatter bar plots quantifying Manders colocalization coefficient between AP-3 with Rab14 and RUFY1-FLAG. Panel L shows confocal microscopy images of control or Arl8b siRNA-treated HeLa cells expressing SBP-GFP-LAMP1 and stained for Rab14. Panel M shows immunoblot images of HeLa cell lysates treated with control or Rab11a siRNA showing Rab11a and α-tubulin protein levels.

Arl8b-positive vesicles transiently interact with AP-3–positive endosomes, which represent the sorting station for the newly synthesized LAMP1 to active lysosomes. (A) HeLa cell lysates treated with control or AP-3 siRNA were IB for the indicated proteins. The values represent densitometric analysis of AP-3 levels normalized to α-tubulin. (B) Representative confocal micrographs of HeLa cells treated with control or AP-3 siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Live-cell imaging was performed after cells were incubated with LTR (magenta) dye to label acidic lysosomes, followed by biotin addition. Yellow arrowheads in the insets denote the colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (C) Manders’ colocalization coefficient quantification of SBP-GFP-LAMP1 with LTR in HeLa cells treated with control or AP-3 siRNA 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 < 0.0001). The values are represented as the mean ± SEM. (D) Bar graph represents the percent MFI of surface LAMP1 in HeLa cells treated with the indicated siRNAs and normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0050). The values are represented as the mean ± SEM. (E) Representative confocal images of HeLa cells treated with control or AP-3 siRNA, followed by co-expression of SBP-GFP-LAMP1 (green) and Arl8b-Halo (magenta). Cells were incubated with biotin, and live-cell imaging was performed. Scale bars: 10 μm (main); 2 μm (inset). (F) Quantification of Manders’ colocalization coefficient of SBP-GFP-LAMP1 with Arl8b-Halo in HeLa cells treated with the 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 (n.s., non-significant). The values are represented as the mean ± SEM. (G) Representative confocal micrographs of Arl8bEN-mStayGold KI (green) HeLa cells immunostained for endogenous AP-3 (magenta). Yellow arrowheads in the insets denote the colocalized pixels. The line profiles indicate fluorescence intensity along the yellow lines for both channels: Arl8bEN-mStayGold (green) and AP-3 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (H) Quantification of Manders’ colocalization coefficient (M1 and M2) of Arl8bEN-mStayGold with AP-3 in HeLa cells 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. The values are represented as the mean ± SEM. (I) Representative confocal micrograph from a live-cell imaging experiment of HeLa cells co-expressing Arl8b-GFP (green) and AP-3 (M1)-mScarlet (magenta); insets show time-lapse images. Micrographs are maximum-intensity projections of z-stack images from a live-cell video. Yellow arrowheads in the insets denote the colocalized pixels. The line profiles indicate fluorescence intensity along the yellow lines for both channels: Arl8b-GFP (green) and AP-3 (M1)-mScarlet (magenta). Scale bars: 10 μm (main); 2 μm (inset). (J) Representative confocal micrographs of HeLa cells immunostained for endogenous AP-3 (magenta) and Rab14 (green) (left panel). Representative confocal images of HeLa cells expressing RUFY1-FLAG were immunostained with anti-FLAG (green) and anti-AP-3 antibodies (magenta) (right panel). Yellow arrowheads in the insets denote the colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (K) Quantification of Manders’ colocalization coefficient of AP-3 with Rab14 and RUFY1-FLAG, respectively, 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. The values are represented as the mean ± SEM. (L) Representative confocal images of HeLa cells treated with control or Arl8b siRNA, followed by the expression of SBP-GFP-LAMP1 (green). Cells were fixed at 60 min after biotin addition and immunostained for endogenous Rab14 (magenta). Scale bars: 10 μm (main); 2 μm (inset). (M) HeLa cell lysates treated with the control or Rab11a siRNAs were IB for the indicated proteins. The values represent densitometric analysis of Rab11a levels normalized to α-tubulin. Source data are available for this figure: SourceData FS3.

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