Figure 5.
ATG9A protein regulates delivery of β1 integrin to cell protrusions. (A) U87 MG cells expressing ATG9A-mCherry were labeled for β1 integrin (green), TGN46 (white), and nuclei (DAPI labeling, blue). Scale bar, 20 µm. Note, from the magnified views on the right panels, that ATG9A-mCherry, β1 integrin, and TGN46 signals partially colocalize in tubular or vesicular structures (arrowheads) near the cell membrane or in the perinuclear area. Scale bar for magnified views, 6 µm. (B) U87 MG cells were transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG9A (siATG9A #1, siATG9A #2). Transfected cells were starved (30 min) in serum-free medium, incubated (30 min) with or without EGF (50 ng/ml), fixed, and labeled for β1 integrin. Upper images: β1 integrin immunoreactive signal (false-color scale) in representative fields of each experimental group. Scale bar, 20 µm. Middle images: Polar distribution of the β1 integrin immunoreactive signal (false-color scale; Clock Scan plug-in) from the cell marked by an asterisk in the upper image. Lower panels: Radial intensity profile of the β1 integrin signal, from the cell centroid to the cell edge (Clock Scan plug-in). For each experimental group, profiles were normalized and averaged from 76–98 cells per group (two independent experiments). (C) Measurement of peripheral β1 integrin signal (signal located in the 80–100% interval of the cell radius, normalized to the total signal) from images shown in B. Cells from independent experiments were color-coded. (D) Validation of ATG5 knockdown. Western blot analysis of ATG5 protein levels from U87 MG cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG5 (siATG5 #1, siATG5 #2). MM, molecular mass. Asterisk, nonspecific band. (E) U87 MG cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG5 (siATG5 #1, siATG5 #2) were incubated with or without EGF, and distribution of the β1 integrin immunoreactive signal was analyzed and presented as in B. For each experimental group, profiles were normalized and averaged from 85–103 cells per group (two independent experiments). Scale bar, 20 µm. (F) Measurement of peripheral β1 integrin signal (signal located in the 80–100% interval of the cell radius, normalized to the total signal) from images shown in E. Cells from independent experiments were color-coded. Data represent means and SEM. Statistical significance was evaluated using one-way ANOVA followed by Tukey post hoc test (C and F). ***, P < 0.001.

ATG9A protein regulates delivery of β1 integrin to cell protrusions. (A) U87 MG cells expressing ATG9A-mCherry were labeled for β1 integrin (green), TGN46 (white), and nuclei (DAPI labeling, blue). Scale bar, 20 µm. Note, from the magnified views on the right panels, that ATG9A-mCherry, β1 integrin, and TGN46 signals partially colocalize in tubular or vesicular structures (arrowheads) near the cell membrane or in the perinuclear area. Scale bar for magnified views, 6 µm. (B) U87 MG cells were transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG9A (siATG9A #1, siATG9A #2). Transfected cells were starved (30 min) in serum-free medium, incubated (30 min) with or without EGF (50 ng/ml), fixed, and labeled for β1 integrin. Upper images: β1 integrin immunoreactive signal (false-color scale) in representative fields of each experimental group. Scale bar, 20 µm. Middle images: Polar distribution of the β1 integrin immunoreactive signal (false-color scale; Clock Scan plug-in) from the cell marked by an asterisk in the upper image. Lower panels: Radial intensity profile of the β1 integrin signal, from the cell centroid to the cell edge (Clock Scan plug-in). For each experimental group, profiles were normalized and averaged from 76–98 cells per group (two independent experiments). (C) Measurement of peripheral β1 integrin signal (signal located in the 80–100% interval of the cell radius, normalized to the total signal) from images shown in B. Cells from independent experiments were color-coded. (D) Validation of ATG5 knockdown. Western blot analysis of ATG5 protein levels from U87 MG cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG5 (siATG5 #1, siATG5 #2). MM, molecular mass. Asterisk, nonspecific band. (E) U87 MG cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG5 (siATG5 #1, siATG5 #2) were incubated with or without EGF, and distribution of the β1 integrin immunoreactive signal was analyzed and presented as in B. For each experimental group, profiles were normalized and averaged from 85–103 cells per group (two independent experiments). Scale bar, 20 µm. (F) Measurement of peripheral β1 integrin signal (signal located in the 80–100% interval of the cell radius, normalized to the total signal) from images shown in E. Cells from independent experiments were color-coded. Data represent means and SEM. Statistical significance was evaluated using one-way ANOVA followed by Tukey post hoc test (C and F). ***, P < 0.001.

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