![Lysosomal cargo degradation is impaired upon TBC1D9B depletion. (A) Graph represents the percent MFI of dequenched BODIPY FL-BSA in HeLa cells treated with control or TBC1D9B siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (*P = 0.0231). The values are represented as the mean ± SEM. (B) Confocal micrographs of HeLa cells treated with the indicated siRNAs and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct, followed by EGF (100 ng/ml) uptake for 2 h. Cells were fixed and immunostained with anti-EGFR (green) and anti-HA (magenta) antibodies. Scale bar: 10 µm. (C) Graph shows quantification of CTCF of EGFR normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0033 [TBC1D9B siRNA]; **P = 0.0042 [RYQ→A]; **P = 0.0034 [E91A]; n.s., non-significant). The values are represented as the mean ± SEM. (D) HeLa cells treated with control or TBC1D9B siRNA were incubated with puromycin (3 μg/ml) for 2 h, followed by a 3-h chase in complete media. Cells were fixed and immunostained with an anti-p62 antibody. Confocal micrographs of the indicated conditions are shown. Scale bar: 10 µm. (E) Quantification of the number of p62-positive punctae per cell for each condition in control or TBC1D9B 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 = 0.0143; n.s., non-significant). The values are represented as the mean ± SEM. 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_fig9.png?Expires=1782456191&Signature=VUsQq0FoJJD5roga5N8PJnjPYJHNSjj3XfqDxTD6-hVzFIpfuKwV7clBvUSaATYdaHhtepHxtDzadvAErbinZ893b9wD8bFawm8Cq8YSMSz~9hWIfWEO7O14hrOlr9vvdr~1fp6QxO2rutowbTJNDhnuCR7ngyX6eagLGv9OFq5qsJnjbErhH4apsXI6q5igBKheKNN~yEtrAiIiXuLPMqrVgEglMS996vqT4GxePnmmi3Js-x-HL4zINfJ3kjEYfz0GDz1kbUoVlH5kUfn7SGfYdDr4OkFXjbLb5-pUjFNmXfbNfIG6sQLOyqOf2INhvs0gNui2iAxICXPp0n7WIQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows a bar graph comparing relative mean fluorescence intensity of dequenched BODIPY FL-BSA in control and TBC1D9B siRNA-treated HeLa cells, indicating lysosomal degradation capacity. Panel B shows confocal microscopy images of HeLa cells treated with control or TBC1D9B siRNA and rescued with HA-TBC1D9B constructs, illustrating EGFR uptake and localization. Panel C shows a bar graph quantifying relative EGFR fluorescence intensity across control, TBC1D9B depletion, and rescue conditions. Panel D shows confocal microscopy images of p62 staining in control and TBC1D9B siRNA-treated HeLa cells under untreated, puromycin-treated, and recovery conditions. Panel E shows scatter bar plots quantifying the number of p62-positive puncta per cell across different treatment conditions.
Lysosomal cargo degradation is impaired upon TBC1D9B depletion. (A) Graph represents the percent MFI of dequenched BODIPY FL-BSA in HeLa cells treated with control or TBC1D9B siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (*P = 0.0231). The values are represented as the mean ± SEM. (B) Confocal micrographs of HeLa cells treated with the indicated siRNAs and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct, followed by EGF (100 ng/ml) uptake for 2 h. Cells were fixed and immunostained with anti-EGFR (green) and anti-HA (magenta) antibodies. Scale bar: 10 µm. (C) Graph shows quantification of CTCF of EGFR normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (**P = 0.0033 [TBC1D9B siRNA]; **P = 0.0042 [RYQ→A]; **P = 0.0034 [E91A]; n.s., non-significant). The values are represented as the mean ± SEM. (D) HeLa cells treated with control or TBC1D9B siRNA were incubated with puromycin (3 μg/ml) for 2 h, followed by a 3-h chase in complete media. Cells were fixed and immunostained with an anti-p62 antibody. Confocal micrographs of the indicated conditions are shown. Scale bar: 10 µm. (E) Quantification of the number of p62-positive punctae per cell for each condition in control or TBC1D9B 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 = 0.0143; n.s., non-significant). The values are represented as the mean ± SEM.