Figure 1. Ub-positive endosomes... | Rockefeller University Press

Figure 1.

$Figure 1. Ub-positive endosomes containing Salmonella or beads are targeted by autophagy. (A) HeLa cells were infected with S. Typhimurium (Salmonella) for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor (TfR). Bar, 10 µm. (B) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3. Bar, 5 µm. (C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student’s unpaired t test. *, P < 0.01. (D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead–autophagosomes were fractionated as described in Materials and methods. The bead–autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized. (F) The bead–autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies. (G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student’s unpaired t test. *, P < 0.05; NS, not significant.$

Ub-positive endosomes containing Salmonella or beads are targeted by autophagy. (A) HeLa cells were infected with S. Typhimurium (Salmonella) for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor (TfR). Bar, 10 µm. (B) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3. Bar, 5 µm. (C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student’s unpaired t test. *, P < 0.01. (D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead–autophagosomes were fractionated as described in Materials and methods. The bead–autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized. (F) The bead–autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies. (G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student’s unpaired t test. *, P < 0.05; NS, not significant.