Figure 6.
Depletion of migfilin does not affect autophagy initiation. (A) KP4 cells were transfected with ULK1-GFP. Representative images of ULK1-GFP puncta were shown in control (siCtrl) or migfilin knockdown (siMig #1) KP4 cells under both control and starvation conditions. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of ULK1-GFP puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 40 cells for each group. (B) KP4 cells were stably transfected with DFCP1-GFP. Representative images of DFCP1-GFP puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of DFCP1-GFP puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 30 cells for each group. (C) KP4 cells were stably transfected with ATG14-GFP. Representative images of ATG14-GFP puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of ATG14-GFP puncta was shown in the lower panel. Data represent mean ± SE, **P < 0.01, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments with at least 34 cells for each group. (D) KP4 cells were stably transfected with GFP-ATG16L. Representative images of GFP-ATG16L puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of GFP-ATG16L puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments with at least 28 cells for each group. (E) KP4 cells were stably transfected with mCherry-Stx17. Representative images of mCherry-Stx17 puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of mCherry-Stx17 puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments with at least 33 cells for each group. (F) Immunoblotting analysis of the protein expression level of total S6K (T-S6K), phosphorylated (p)-S6K, total S6 (T-S6), phosphorylated (p)-S6, total 4EBP1 (T-4EBP1), phosphorylated (p)-4EBP1 in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control condition. Quantification analysis of the ratios of p-S6K/T-S6K, p-S6/T-S6, and p-4EBP1/T-4EBP1 was shown in the lower panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 6 independent experiments. Source data are available for this figure: SourceData F6.

Depletion of migfilin does not affect autophagy initiation. (A) KP4 cells were transfected with ULK1-GFP. Representative images of ULK1-GFP puncta were shown in control (siCtrl) or migfilin knockdown (siMig #1) KP4 cells under both control and starvation conditions. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of ULK1-GFP puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 40 cells for each group. (B) KP4 cells were stably transfected with DFCP1-GFP. Representative images of DFCP1-GFP puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of DFCP1-GFP puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 30 cells for each group. (C) KP4 cells were stably transfected with ATG14-GFP. Representative images of ATG14-GFP puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of ATG14-GFP puncta was shown in the lower panel. Data represent mean ± SE, **P < 0.01, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments with at least 34 cells for each group. (D) KP4 cells were stably transfected with GFP-ATG16L. Representative images of GFP-ATG16L puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of GFP-ATG16L puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments with at least 28 cells for each group. (E) KP4 cells were stably transfected with mCherry-Stx17. Representative images of mCherry-Stx17 puncta were shown. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of mCherry-Stx17 puncta was shown in the lower panel. Data represent mean ± SE, ***P < 0.001, unpaired two-tailed t test. n = 3 independent experiments with at least 33 cells for each group. (F) Immunoblotting analysis of the protein expression level of total S6K (T-S6K), phosphorylated (p)-S6K, total S6 (T-S6), phosphorylated (p)-S6, total 4EBP1 (T-4EBP1), phosphorylated (p)-4EBP1 in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control condition. Quantification analysis of the ratios of p-S6K/T-S6K, p-S6/T-S6, and p-4EBP1/T-4EBP1 was shown in the lower panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 6 independent experiments. Source data are available for this figure: SourceData F6.

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