![TBC1D9B depletion alters lysosome characteristics and impairs CI-M6PR retrieval to the TGN, compromising cathepsin processing. (A) Representative confocal micrographs of HeLa cells treated with control or TBC1D9B siRNA and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct. Cells were fixed and immunostained with anti-HA (green) and anti-LAMP1 (magenta) antibodies. Scale bar: 10 μm. (B) Quantification of the perinuclear index of LAMP1-positive compartments in HeLa cells treated with control or TBC1D9B siRNA and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct 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 = 0.0003; **P = 0.0013 (RYQ→A); **P = 0.0023 (E91A); n.s., non-significant). The values are represented as the mean ± SEM. (C) Representative confocal images of HeLa cells treated with control or TBC1D9B siRNA and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct. Cells were fixed and immunostained with anti-HA (magenta) and anti-cathepsin D (green) antibodies. Scale bar: 10 μm. (D) The graph shows the relative percentage of CTCF values of the cathepsin D signal normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (***P = 0.0003 (TBC1D9B siRNA and RYQ→A); ***P = 0.0001 (E91A); n.s., non-significant). The values are represented as the mean ± SEM. (E) Representative confocal images of HeLa cells treated with control or TBC1D9B siRNA and immunostained with anti-LAMP1 (green) and anti-cathepsin D (magenta) antibodies. Scale bars: 10 μm (main); 2 μm (inset). (F) Manders’ colocalization coefficient quantification of LAMP1 with cathepsin D in HeLa cells treated with the indicated siRNAs 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 = 0.0008). The values are represented as the mean ± SEM. (G) Western blot analysis of cathepsin D levels in lysates and the culture supernatant was performed in control and TBC1D9B siRNA-treated HeLa cells. The Ponceau S stain was done to visualize protein levels. The values represent densitometric analysis of cathepsin D levels normalized to α-tubulin (lysate) or total protein (media). (H) Representative confocal micrographs of HeLa cells treated with control or TBC1D9B siRNA and costained for CI-M6PR (magenta) and Giantin (green) or Rab14 (green) or Rab11a (green). The yellow arrowheads denote colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (I) Quantification of Manders’ colocalization coefficient of CI-M6PR with Giantin, Rab14, or Rab11a 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 (**P = 0.0029 (Giantin); **P = 0.0014 [Rab14]; ***P = 0.0001 [Rab11a]). The values are represented as the mean ± SEM. CTCF, corrected total cell fluorescence. Source data are available for this figure: SourceData F8. 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_fig8.png?Expires=1782456305&Signature=LxbDt0gYWRIfvgvwcq5lz4FjBArZPXpOOqzYM~cMSMhvcWK1ejEjdb6aAgUcFUVry1738B87Of682-2akmW206equhPGAUUMwUPujaui8m~5HP~OsoqAJjrUMhGIGmup~hUnH55oUUNRqpQcANSy6VVMytFzeMS6q1gkncYGeulwoXsCvrNDMAFz0ChZEQcJG9kMt463hX7DqROT726ASf5p8OobNpNXjVzfjqwcgumW8FciJWglYQ4ePhw5cE-0mB3mC9aK~eKkCiLBjlCQT0Ji6eGa~bq0dFyK65B00plyLjptx6rNn7lioQskMmUUZC3B3CSSXxVczogxYVvdog__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows confocal microscopy images of HeLa cells treated with control or TBC1D9B siRNA and rescued with HA-TBC1D9B constructs, highlighting LAMP1 distribution patterns. Panel B shows scatter bar plot quantifying the perinuclear index of LAMP1-positive compartments across control, knockdown, and rescue conditions. Panel C shows confocal microscopy images of HeLa cells stained for cathepsin D under control, TBC1D9B depletion, and rescue conditions. Panel D shows a bar graph quantifying relative cathepsin D fluorescence intensity across experimental conditions. Panel E shows confocal microscopy images of LAMP1 and cathepsin D localization in control and TBC1D9B siRNA-treated cells with magnified views. Panel F shows scatter bar plot quantifying Manders colocalization coefficient between LAMP1 and cathepsin D in control and TBC1D9B siRNA-treated cells. Panel G shows immunoblot images analyzing pro and mature cathepsin D levels in cell lysates and culture supernatants under control and TBC1D9B depletion conditions. Panel H shows confocal microscopy images of CI-M6PR localization relative to Giantin, Rab14, and Rab11a in control and TBC1D9B siRNA-treated cells. Panel I shows scatter dot plots quantifying Manders colocalization coefficient between CI-M6PR and indicated markers under control and TBC1D9B depletion conditions.
TBC1D9B depletion alters lysosome characteristics and impairs CI-M6PR retrieval to the TGN, compromising cathepsin processing. (A) Representative confocal micrographs of HeLa cells treated with control or TBC1D9B siRNA and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct. Cells were fixed and immunostained with anti-HA (green) and anti-LAMP1 (magenta) antibodies. Scale bar: 10 μm. (B) Quantification of the perinuclear index of LAMP1-positive compartments in HeLa cells treated with control or TBC1D9B siRNA and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct 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 = 0.0003; **P = 0.0013 (RYQ→A); **P = 0.0023 (E91A); n.s., non-significant). The values are represented as the mean ± SEM. (C) Representative confocal images of HeLa cells treated with control or TBC1D9B siRNA and transfected with the HA-TBC1D9B (WT, RYQ→A, or E91A rescue) construct. Cells were fixed and immunostained with anti-HA (magenta) and anti-cathepsin D (green) antibodies. Scale bar: 10 μm. (D) The graph shows the relative percentage of CTCF values of the cathepsin D signal normalized to control siRNA (n = 3; each dot represents a single experiment). Statistical significance was calculated using the one-sample t test (***P = 0.0003 (TBC1D9B siRNA and RYQ→A); ***P = 0.0001 (E91A); n.s., non-significant). The values are represented as the mean ± SEM. (E) Representative confocal images of HeLa cells treated with control or TBC1D9B siRNA and immunostained with anti-LAMP1 (green) and anti-cathepsin D (magenta) antibodies. Scale bars: 10 μm (main); 2 μm (inset). (F) Manders’ colocalization coefficient quantification of LAMP1 with cathepsin D in HeLa cells treated with the indicated siRNAs 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 = 0.0008). The values are represented as the mean ± SEM. (G) Western blot analysis of cathepsin D levels in lysates and the culture supernatant was performed in control and TBC1D9B siRNA-treated HeLa cells. The Ponceau S stain was done to visualize protein levels. The values represent densitometric analysis of cathepsin D levels normalized to α-tubulin (lysate) or total protein (media). (H) Representative confocal micrographs of HeLa cells treated with control or TBC1D9B siRNA and costained for CI-M6PR (magenta) and Giantin (green) or Rab14 (green) or Rab11a (green). The yellow arrowheads denote colocalized pixels. Scale bars: 10 μm (main); 2 μm (inset). (I) Quantification of Manders’ colocalization coefficient of CI-M6PR with Giantin, Rab14, or Rab11a 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 (**P = 0.0029 (Giantin); **P = 0.0014 [Rab14]; ***P = 0.0001 [Rab11a]). The values are represented as the mean ± SEM. CTCF, corrected total cell fluorescence. Source data are available for this figure: SourceData F8.