![TBC1D9B depletion leads to an increase in surface LAMP1 levels and alters the number, size, and distribution of lysosomes. (A) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with indicated markers 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. (B) Lysates of HeLa, HEK293T, RPE-1, and THP-1 cells treated with control or TBC1D9B siRNA were IB with the indicated antibodies. The values indicate densitometric analysis of TBC1D9B levels normalized to α-tubulin. The bar graph represents the relative fold change in densitometric values of TBC1D9B normalized to α-tubulin. Statistical significance was calculated using the one-sample t test (n = 3; **P = 0.0011 [HeLa]; ***P = 0.0004 [HEK293T]; ***P = 0.0007 pRPE-1]; **P = 0.007 [THP-1]). The values are represented as the mean ± SEM. (C) Bar graph represents the percent MFI of surface EGFR levels in HeLa cells treated with indicated siRNAs 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) Graph represents the MFI of surface LAMP1 levels normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (n.s., non-significant). The values are represented as the mean ± SEM. (E) Representative immuno-EM images of HeLa cells treated with control or TBC1D9B siRNA and immunolabeled for LAMP1 (15 nm). Higher magnifications of LAMP1-positive vesicles are shown in the panels on the right. Scale bars: 200 nm (main); 100 nm (inset). (F) Lysosome diameter was quantified from immuno-EM images of HeLa cells treated with the indicated siRNAs (n = 1; 14 and 16 micrographs of control siRNA and TBC1D9B siRNA-treated cells, respectively). The values are represented as the mean. (G–J) HeLa cells treated with control or TBC1D9B siRNA were incubated overnight with Alexa Fluor 568–conjugated dextran, followed by a 6-h chase to label lysosomes, and live-cell imaging was performed. The graph (G) represents the mean lysosome number per cell in control or TBC1D9B-depleted cells analyzed from two independent experiments (n = 2; each dot represents a single cell). The values are represented as the mean. The graph (H) represents the mean lysosome area of peripheral and perinuclear lysosomes in control or TBC1D9B siRNA-treated HeLa cells analyzed from two independent experiments (n = 2; each dot represents a single cell). The values are represented as the mean. The graph (I) represents Ripley’s constant analysis. The green line depicts random distribution, whereas the blue and red lines denote control and TBC1D9B siRNA-treated samples, respectively. The graph (J) represents the diffusion exponent of dextran-loaded terminal lysosomes in control or TBC1D9B siRNA-treated cells and analyzed from two independent experiments (n = 2; each dot represents a single cell). The values are represented as the mean. (K) Kinetic analysis of GTPase activity for Rab5a, Rab11a, and Rab14 in the presence of TBC1D9B (WT) or its GAP-deficient mutant (RYQ→A). The plotted values were calculated by subtracting the signal of GDP-loaded samples from GTP-loaded samples. Source data are available for this figure: SourceData FS5. 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_figs5.png?Expires=1782456202&Signature=ifWw4X85ZoDuBpucmyS2wbNkZKuBWma3SeKeO9nzM4JLrIi6mr7pEMz0UfXPYaVugXcLNxxPbIPIe3HG35IS~a3XAWIPLel~WT-XT~Tjft0e9oKRgPIUZdg4g7O8vl9zNJ89mRgzy1TNbwUm23dqBDQpAj7w4EHkQemSnuchrS~z0LLpDPg5cCu8ntpIJsf8RyU4CMetgPGE6scMOAbejP6inGIjLjlCi77nJY69dt-BTxAubv~apEIl4x1Jq55pJ-7nXeWzFucvnxg4fu1UhdRBrm9QoJ8RQboPomKrQ7Zxn1K5CKl~pr-~Aap6nfdYWYj7Q9mlunA7su4vTVQvHg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows scatter bar plot quantifying Manders colocalization coefficient of GFP-TBC1D9B with RUFY1-Halo and Halo-Rab14 across individual cells. Panel B shows immunoblot images and bar graph comparing TBC1D9B protein levels in multiple cell lines treated with control or TBC1D9B siRNA. Panel C shows a bar graph comparing relative mean fluorescence intensity of surface EGFR in control and TBC1D9B siRNA-treated HeLa cells. Panel D shows bar graph comparing relative mean fluorescence intensity of surface LAMP1 in RPE-1, THP-1, and HEK293T cells treated with control or TBC1D9B siRNA. Panel E shows immuno-electron microscopy images of LAMP1-labeled vesicles in control and TBC1D9B siRNA-treated HeLa cells with magnified views. Panel F shows scatter bar plot quantifying lysosome diameter in control and TBC1D9B siRNA-treated cells based on electron microscopy analysis. Panel G shows scatter bar plot comparing mean lysosome number per cell between control and TBC1D9B siRNA-treated conditions. Panel H shows scatter bar plot comparing mean lysosome area in peripheral and perinuclear regions for control and TBC1D9B siRNA-treated cells. Panel I shows line graph representing Ripley’s K function analysis of lysosome spatial distribution in control and TBC1D9B siRNA-treated cells. Panel J shows scatter bar plot comparing diffusion exponent of lysosomes in peripheral, perinuclear, and whole-cell regions under control and TBC1D9B siRNA conditions. Panel K shows line graph depicting GTPase activity kinetics of Rab5a, Rab11a, and Rab14 in the presence of TBC1D9B wild-type or GAP-deficient mutant.
TBC1D9B depletion leads to an increase in surface LAMP1 levels and alters the number, size, and distribution of lysosomes. (A) Quantification of Manders’ colocalization coefficient of GFP-TBC1D9B with indicated markers 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. (B) Lysates of HeLa, HEK293T, RPE-1, and THP-1 cells treated with control or TBC1D9B siRNA were IB with the indicated antibodies. The values indicate densitometric analysis of TBC1D9B levels normalized to α-tubulin. The bar graph represents the relative fold change in densitometric values of TBC1D9B normalized to α-tubulin. Statistical significance was calculated using the one-sample t test (n = 3; **P = 0.0011 [HeLa]; ***P = 0.0004 [HEK293T]; ***P = 0.0007 pRPE-1]; **P = 0.007 [THP-1]). The values are represented as the mean ± SEM. (C) Bar graph represents the percent MFI of surface EGFR levels in HeLa cells treated with indicated siRNAs 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) Graph represents the MFI of surface LAMP1 levels normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (n.s., non-significant). The values are represented as the mean ± SEM. (E) Representative immuno-EM images of HeLa cells treated with control or TBC1D9B siRNA and immunolabeled for LAMP1 (15 nm). Higher magnifications of LAMP1-positive vesicles are shown in the panels on the right. Scale bars: 200 nm (main); 100 nm (inset). (F) Lysosome diameter was quantified from immuno-EM images of HeLa cells treated with the indicated siRNAs (n = 1; 14 and 16 micrographs of control siRNA and TBC1D9B siRNA-treated cells, respectively). The values are represented as the mean. (G–J) HeLa cells treated with control or TBC1D9B siRNA were incubated overnight with Alexa Fluor 568–conjugated dextran, followed by a 6-h chase to label lysosomes, and live-cell imaging was performed. The graph (G) represents the mean lysosome number per cell in control or TBC1D9B-depleted cells analyzed from two independent experiments (n = 2; each dot represents a single cell). The values are represented as the mean. The graph (H) represents the mean lysosome area of peripheral and perinuclear lysosomes in control or TBC1D9B siRNA-treated HeLa cells analyzed from two independent experiments (n = 2; each dot represents a single cell). The values are represented as the mean. The graph (I) represents Ripley’s constant analysis. The green line depicts random distribution, whereas the blue and red lines denote control and TBC1D9B siRNA-treated samples, respectively. The graph (J) represents the diffusion exponent of dextran-loaded terminal lysosomes in control or TBC1D9B siRNA-treated cells and analyzed from two independent experiments (n = 2; each dot represents a single cell). The values are represented as the mean. (K) Kinetic analysis of GTPase activity for Rab5a, Rab11a, and Rab14 in the presence of TBC1D9B (WT) or its GAP-deficient mutant (RYQ→A). The plotted values were calculated by subtracting the signal of GDP-loaded samples from GTP-loaded samples. Source data are available for this figure: SourceData FS5.