Fig. 5: IGF2R maintains autophagy and mitophagy through lysosome homeostasis. | Cell Death & Disease

Fig. 5: IGF2R maintains autophagy and mitophagy through lysosome homeostasis.

From: Upregulation of IGF2R evades lysosomal dysfunction-induced apoptosis of cervical cancer cells via transport of cathepsins

Fig. 5

a Distribution of IGF2R in subcellular compartments. GM130, Rab5, Rab7, and LAMP1 are markers for the Golgi apparatus, early endosome, late endosome, and lysosome, respectively. b Accumulation of acidic organelles in IGF2R-knockdown cells at 96 h after siRNA transfection. The LysoTracker+ area in the cells was measured and normalized by the number of nuclei. c Activities of lysosomal enzymes in IGF2R-knockdown cells. The activities were assessed using a self-quenching fluorophore-labeled peptide probe, which yields green fluorescence in response to lysosomal enzyme cleavage. Flow cytometric analysis of the probe-treated cells at 96 h after siRNA transfection. d Induction of autophagosome formation by loss of IGF2R. Western blot analysis of the LC3-I and LC3-II status in cells at 96 h after siRNA transfection (left panel). Immunocytochemical analysis of autophagosomes and autolysosomes in the cells under the same conditions (right panel). Anti-LC3 and anti-LAMP1 antibodies were used to detect autophagosomes (green) and lysosomes (red), respectively. e Effect of autophagic flux on IGF2R knockdown. Western blot analysis shows the conversion of LC3-I to LC3-II in cells with siRNA transfection (96 h), followed by treatment with an inhibitor of lysosome acidification, Bafilomycin A1 for 24 h (left panel). Densitometry analysis from three independent experiments (right panel). f Western blot analysis of ubiquitinylated proteins in IGF2R-knockdown cells at 72 h after siRNA transfection. g Reactive oxygen species (ROS) production in cells with IGF2R knockdown. The amount of intracellular ROS was analyzed by CellROX reagent, which yields fluorescence by oxidation. Representative histogram of flow cytometry (top panel) and its mean fluorescent intensity (bottom panel). h Activities of mitophagy in cells with a loss of IGF2R. Representative images of cells under knockdown of IGF2R at 96 h after transfection. Mitochondria and lysosomes in the cells were stained by anti-COX IV (green) and anti-LAMP1 (red) antibodies, respectively. All the error bars represent the standard deviation of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; t-test with Welch’s correction and Tukey multiple comparisons test. Scale bars represent 20 μm.

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