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. 2022 Feb 18;221(3):e202106014. doi: 10.1083/jcb.202106014

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© 2022 Campisi et al.

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Figure 2. — Depletion of ATG9A protein impairs the dynamics of cell protrusions and intrinsic cell speed. (A) Representative images from phase-contrast time-lapse sequences acquired from U87 MG cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG9A (siATG9A #1, siATG9A #2). (B) Percentage of the cell perimeter occupied by ruffling protrusions (n = 94–106 cells per group; cells from independent experiments were color-coded). (C) Phase-contrast time-lapse sequences acquired from U87 MG cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG9A (siATG9A #1 or siATG9A #2). Upper panels: Single images from representative sequences of U87 MG cells transfected with nontargeting siRNA (siCont) and siATG9A #2 (upper left; scale bar, 20 µm), and kymographs (upper right) generated from 1-pixel-wide lines drawn on the cell edges. The scale bars in the kymographs are 20 µm (horizontal) and 40 min (vertical). Lower panels: Quantification, from kymographs, of protrusion velocity (left), protrusion distance (middle), and protrusion persistence (right; n = 84–87 protrusions per group; cells from independent experiments were color-coded). (D) Cell trajectories over a 3-h period (one frame every 40 s) of randomly migrating U87 MG cells transfected with nontargeting siRNA (siCont, n = 101 cells) or siRNA targeting ATG9A (siATG9A #1, n = 88 cells; siATG9A #2, n = 97 cells). (E and F) Analysis of average cell speed (E) and mean square displacement (MSD; F), calculated from the cell trajectories presented in D. (G) Relative ATP levels from lysates of serial amounts of U87 MG cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG9A (siATG9A #1, siATG9A #2). Analyses (n = 3 wells for each cell number) were performed at 24 h (left panel) or 48 h (right panel) after transfection. Results were normalized to that of cells transfected with nontargeting siRNA (2,500 cells). (H) Measurement of cell density at 24 and 48 h after transfection, from cells transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG9A (siATG9A #1, siATG9A #2). Data represent means and SEM. Statistical significance was evaluated using one-way ANOVA followed by Tukey post hoc test (B, C, and E), two-way ANOVA followed by Dunnett post hoc test (F), two-way ANOVA followed by Bonferroni post hoc test (G), or one-way ANOVA followed by Sidak post hoc test (H). ***, P < 0.001.