Figure S2.
Migfilin depletion blocks autophagic flux and inhibits cell growth in human breast cancer cells. (A) Immunoblotting analysis of p62 and LC3 levels in control (siCtrl) and migfilin knockdown (siMig #1) BT549 cells under nutrient-rich (control), EBSS starvation (Starv.) condition. (B and C) Quantification analysis of p62 (B) and LC3-II (C) levels in A. Data represent mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t test. n = 4 independent experiments. (D and E) BT549 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. Representative images were shown in D and quantification analysis was shown in E. Scale bar: 10 µm (Magnify: 2 µm). At least 26 cells in each group were analyzed. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments. (F) BT549 cells were stably transfected with mRFP-GFP-LC3 report system. Representative images of mRFP-LC3 puncta and GFP-LC3 puncta in control (siCtrl) or migfilin knockdown (siMig #1) BT549 cells under control and starvation conditions. Scale bar, 10 µm (Magnify, 2 µm). (G) Quantification of the percentage of RFP+GFP+ puncta to total puncta in F was shown. Data represent mean ± SE, *P < 0.05, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments. At least 30 cells in each experiment were analyzed. (H) BT549 cell lysates were immunoprecipitated (IPed) with anti-migfilin antibody or control IgG (mIgG) followed by immunoblotting with antibodies as indicated. The presence of target proteins in cell lysates was shown as input. (I) Depletion of migfilin in BT549 cells led to a significant decrease in anchorage-dependent colony-forming abilities. Representative images (left panel) and quantification analysis (right panel) were shown. Data represent mean ± SE, ***P < 0.001, n.s., no significance, one-way ANOVA, n = 3 independent experiments. Source data are available for this figure: SourceData FS2.

Migfilin depletion blocks autophagic flux and inhibits cell growth in human breast cancer cells. (A) Immunoblotting analysis of p62 and LC3 levels in control (siCtrl) and migfilin knockdown (siMig #1) BT549 cells under nutrient-rich (control), EBSS starvation (Starv.) condition. (B and C) Quantification analysis of p62 (B) and LC3-II (C) levels in A. Data represent mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t test. n = 4 independent experiments. (D and E) BT549 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. Representative images were shown in D and quantification analysis was shown in E. Scale bar: 10 µm (Magnify: 2 µm). At least 26 cells in each group were analyzed. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments. (F) BT549 cells were stably transfected with mRFP-GFP-LC3 report system. Representative images of mRFP-LC3 puncta and GFP-LC3 puncta in control (siCtrl) or migfilin knockdown (siMig #1) BT549 cells under control and starvation conditions. Scale bar, 10 µm (Magnify, 2 µm). (G) Quantification of the percentage of RFP+GFP+ puncta to total puncta in F was shown. Data represent mean ± SE, *P < 0.05, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments. At least 30 cells in each experiment were analyzed. (H) BT549 cell lysates were immunoprecipitated (IPed) with anti-migfilin antibody or control IgG (mIgG) followed by immunoblotting with antibodies as indicated. The presence of target proteins in cell lysates was shown as input. (I) Depletion of migfilin in BT549 cells led to a significant decrease in anchorage-dependent colony-forming abilities. Representative images (left panel) and quantification analysis (right panel) were shown. Data represent mean ± SE, ***P < 0.001, n.s., no significance, one-way ANOVA, n = 3 independent experiments. Source data are available for this figure: SourceData FS2.

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