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

Figure 1.

Figure 1.

Depletion of ATG9A impairs chemotactic migration and the formation of F-actin–rich protrusions. (A) Effect of siRNA knockdown of ATG9A on chemotactic migration induced by EGF. HeLa cells (left), U87 MG cells (middle), and 42 MG cells (right) were transfected with nontargeting siRNA (siCont) or one of the two siRNAs targeting ATG9A (siATG9A #1, siATG9A #2). Transfected cells were loaded in the upper chamber of Transwells, with or without EGF (50 ng/ml) in the upper or lower chamber, as indicated. After 24 h, cells that migrated onto the lower surface of the membrane were fixed, stained, and counted. Data represent means and SEM (n = 6 Transwells). (B) Effect of siRNA knockdown of ATG9A on chemotactic migration induced by CXCL12. Experiment was performed as described in C, using CXCL12 (10−8 M), on HeLa cells (left), HEK 293 cells (middle), and 42 MG cells (right). (C) Representative epifluorescence images showing F-actin (rhodamine phalloidin labeling; gray levels) and nuclei (DAPI labeling; blue) of U87 MG cells transfected with the indicated siRNAs. Control cells generally develop large F-actin–rich lamellipodia (arrowheads), whereas ATG9A-depleted cells display smaller and irregular protrusions (arrowheads). Scale bars, 20 µm; magnified views, 5 µm. (D) Quantification of F-actin intensities, after background subtraction, in protrusions of U87 MG cells transfected with the indicated siRNAs. For each cell, values correspond to cumulated signal of all protrusions. Data represent means and SEM (n = 106–135 cells per group; cells from independent experiments were color-coded). (E) Percentage of the cell perimeter containing F-actin–rich protrusions, in U87 MG cells transfected with the indicated siRNAs. Data represent means and SEM (n = 209–219 cells per group; cells from independent experiments were color-coded). (F) Effect of siRNA knockdown of ATG9A on cell polarization of U87 MG cells (n = 212–226 cells per group; from three independent experiments). Statistical significance was evaluated using a one-way ANOVA followed by Tukey post hoc test. *, P < 0.05; ***, P < 0.001.