Figure 1.
Migfilin depletion promotes autophagosome accumulation. (A) Immunoblotting analysis of p62 and LC3 levels in control (siCtrl) and migfilin knockdown (siMig #1). KP4 cells under nutrient-rich (control), EBSS starvation (Starv.), or bafilomycin A1 (Baf-A1, 1 μM) treatment condition. (B and C) Quantification analysis of LC3-II (B) and p62 (C) levels in A. Data represent mean ± SE, *P < 0.05, **P < 0.01. n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments. (D) KP4 cells were stably transfected with GFP-LC3 (green) under control or EBSS starvation conditions and imaged by fluorescence microscopy. The number of GFP-LC3 puncta was measured. Representative images were shown in the upper panel and quantification analysis was shown in the lower panel. Scale bar: 10 µm (Magnify: 2 µm). At least 30 images in each group were analyzed. Data represent mean ± SE, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. (E) SW1990 cells were stably transfected with GFP-LC3 (green) under control or EBSS starvation condition and imaged by fluorescence microscopy. The number of GFP-LC3 puncta was measured. Scale bar: 10 µm (Magnify: 2 µm). Representative images were shown in the upper panel and quantification analysis was shown in the lower panel. At least 30 images in each group were analyzed. Data represent mean ± SE, **P < 0.01, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments. (F) Transmission electron microscopy (TEM) analysis of autophagosomes (AP, solid arrows) and autolysosomes (AL, open arrows) in control (siCtrl) or migfilin knockdown (siMig #1) KP4 cells with 1 μM Baf-A1 treatment. Higher magnification of TEM images was shown. Scale bar: 2 µm (left panel), 0.5 µm (right panel). Quantification analysis was shown in the right panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 6 independent experiments. (G) TEM analysis of autophagosomes (solid arrows) and autolysosomes (open arrows) in control (vector) or migfilin overexpressed (Migfilin) KP4 cells under EBSS starvation condition. Higher magnification was shown. Scale bar: 2 µm (left panel), 0.5 µm (right panel). Quantification analysis is shown in the right panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 15 independent experiments. Source data are available for this figure: SourceData F1.

Migfilin depletion promotes autophagosome accumulation. (A) Immunoblotting analysis of p62 and LC3 levels in control (siCtrl) and migfilin knockdown (siMig #1). KP4 cells under nutrient-rich (control), EBSS starvation (Starv.), or bafilomycin A1 (Baf-A1, 1 μM) treatment condition. (B and C) Quantification analysis of LC3-II (B) and p62 (C) levels in A. Data represent mean ± SE, *P < 0.05, **P < 0.01. n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments. (D) KP4 cells were stably transfected with GFP-LC3 (green) under control or EBSS starvation conditions and imaged by fluorescence microscopy. The number of GFP-LC3 puncta was measured. Representative images were shown in the upper panel and quantification analysis was shown in the lower panel. Scale bar: 10 µm (Magnify: 2 µm). At least 30 images in each group were analyzed. Data represent mean ± SE, ***P < 0.001, one-way ANOVA. n = 3 independent experiments. (E) SW1990 cells were stably transfected with GFP-LC3 (green) under control or EBSS starvation condition and imaged by fluorescence microscopy. The number of GFP-LC3 puncta was measured. Scale bar: 10 µm (Magnify: 2 µm). Representative images were shown in the upper panel and quantification analysis was shown in the lower panel. At least 30 images in each group were analyzed. Data represent mean ± SE, **P < 0.01, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments. (F) Transmission electron microscopy (TEM) analysis of autophagosomes (AP, solid arrows) and autolysosomes (AL, open arrows) in control (siCtrl) or migfilin knockdown (siMig #1) KP4 cells with 1 μM Baf-A1 treatment. Higher magnification of TEM images was shown. Scale bar: 2 µm (left panel), 0.5 µm (right panel). Quantification analysis was shown in the right panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 6 independent experiments. (G) TEM analysis of autophagosomes (solid arrows) and autolysosomes (open arrows) in control (vector) or migfilin overexpressed (Migfilin) KP4 cells under EBSS starvation condition. Higher magnification was shown. Scale bar: 2 µm (left panel), 0.5 µm (right panel). Quantification analysis is shown in the right panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 15 independent experiments. Source data are available for this figure: SourceData F1.

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