Figure S4.
ATG9A protein regulates delivery of TGN46-positive post-Golgi carriers to cell protrusions. (A) Left: Representative U87 MG cell expressing ATG9A-mCherry (red) and colabeled for TGN46 (green) and nuclei (DAPI labeling, blue). Scale bar, 20 µm. Right: Magnified views of the cell presented on the left. Note that ATG9A colocalizes with TGN46 in typical TGN cisternae (upper panels) and at the cell membrane or vesicles close to the cell membrane (lower panels). Scale bar for magnified views, 5 µm. (B–E) Chemotactic stimulation induces redistribution of TGN46 and ATG9A-mCherry toward the cell membrane. (B) U87 MG cells expressing ATG9A-mCherry were starved (30 min) in serum-free medium and incubated (30 min) with or without EGF (50 ng/ml), as indicated. Cells were fixed and labeled for TGN46 (green) and nuclei (DAPI labeling, blue). Scale bar, 20 µm. (C) Quantification from images shown in B of the TGN46 and ATG9A-mCherry signals located in protrusions. For each cell, values correspond to the cumulated signal of all protrusions. (D) Quantification from images shown in B of the TGN46 and ATG9A-mCherry signals located in the perinuclear area. Data represent means ± SEM (n = 68 cells per group; cells from independent experiments were color-coded). (E) Cell-to-cell correlation between TGN46 and ATG9A-mCherry signals located in cell protrusions, in both untreated (black dots) and EGF-treated (red dots) cells. (F–H) EGF-induced redistribution of TGN46 to the cell protrusions depends on ATG9A. (F) U87 MG cells were transfected with nontargeting siRNA (siCont) or one of the two siRNA targeting ATG9A (siATG9A #1, siATG9A #2). Transfected cells were starved (30 min) in serum-free medium and incubated (30 min) with or without EGF (50 ng/ml), as indicated. Cells were fixed and labeled for TGN46 (green) and nuclei (DAPI labeling, blue). Scale bar, 20 µm. (G) Quantification from images shown in F of the TGN46 signal located in protrusions. (H) Quantification from images shown in F of the TGN46 signal located in the perinuclear area. Data represent means ± SEM (n = 104–113 cells per group; cells from independent experiments were color-coded). (I) Detection (left) and quantification (right), from cells treated as in F, of the number of cytosolic TGN46-positive vesicles. Data represent means ± SEM (n = 99–112 cells per group; cells from independent experiments were color-coded). Statistical significance was evaluated using Mann–Whitney U test (C and D), one-way ANOVA followed by Tukey post hoc test (G and I), or one-way ANOVA followed by Sidak post hoc test (H), **, P < 0.01; ***, P < 0.001.

ATG9A protein regulates delivery of TGN46-positive post-Golgi carriers to cell protrusions. (A) Left: Representative U87 MG cell expressing ATG9A-mCherry (red) and colabeled for TGN46 (green) and nuclei (DAPI labeling, blue). Scale bar, 20 µm. Right: Magnified views of the cell presented on the left. Note that ATG9A colocalizes with TGN46 in typical TGN cisternae (upper panels) and at the cell membrane or vesicles close to the cell membrane (lower panels). Scale bar for magnified views, 5 µm. (B–E) Chemotactic stimulation induces redistribution of TGN46 and ATG9A-mCherry toward the cell membrane. (B) U87 MG cells expressing ATG9A-mCherry were starved (30 min) in serum-free medium and incubated (30 min) with or without EGF (50 ng/ml), as indicated. Cells were fixed and labeled for TGN46 (green) and nuclei (DAPI labeling, blue). Scale bar, 20 µm. (C) Quantification from images shown in B of the TGN46 and ATG9A-mCherry signals located in protrusions. For each cell, values correspond to the cumulated signal of all protrusions. (D) Quantification from images shown in B of the TGN46 and ATG9A-mCherry signals located in the perinuclear area. Data represent means ± SEM (n = 68 cells per group; cells from independent experiments were color-coded). (E) Cell-to-cell correlation between TGN46 and ATG9A-mCherry signals located in cell protrusions, in both untreated (black dots) and EGF-treated (red dots) cells. (F–H) EGF-induced redistribution of TGN46 to the cell protrusions depends on ATG9A. (F) U87 MG cells were transfected with nontargeting siRNA (siCont) or one of the two siRNA targeting ATG9A (siATG9A #1, siATG9A #2). Transfected cells were starved (30 min) in serum-free medium and incubated (30 min) with or without EGF (50 ng/ml), as indicated. Cells were fixed and labeled for TGN46 (green) and nuclei (DAPI labeling, blue). Scale bar, 20 µm. (G) Quantification from images shown in F of the TGN46 signal located in protrusions. (H) Quantification from images shown in F of the TGN46 signal located in the perinuclear area. Data represent means ± SEM (n = 104–113 cells per group; cells from independent experiments were color-coded). (I) Detection (left) and quantification (right), from cells treated as in F, of the number of cytosolic TGN46-positive vesicles. Data represent means ± SEM (n = 99–112 cells per group; cells from independent experiments were color-coded). Statistical significance was evaluated using Mann–Whitney U test (C and D), one-way ANOVA followed by Tukey post hoc test (G and I), or one-way ANOVA followed by Sidak post hoc test (H), **, P < 0.01; ***, P < 0.001.

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