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. 2023 Jun 20;222(9):e202208047. doi: 10.1083/jcb.202208047

Figure 4.

Figure 4.

Involvement of the PI3K/Akt signaling pathway in Polydom-induced LEC migration. (A) LECs were allowed to migrate to the lower side of Transwell membranes in the absence (none) or presence of Polydom (1 μg/ml) with an ERK inhibitor (2 μM SCH772984) or PI3K inhibitors (20 μM LY294002 or 2 μM Wortmannin) added to the lower chamber medium. Cells that migrated to the lower side of the membranes were counted under a microscope. The data represent means ± SD of three independent experiments each assayed in triplicate. NS, not significant; **P < 0.01 (n = 3). (B) Serum-starved LECs were treated with Polydom (1 μg/ml), Polydom/E2567A (1 μg/ml), COMP-Ang1 (500 ng/ml), or Ang1 (500 ng/ml) in medium containing 0.5% FBS at 37°C for 15 min. Total lysates of the cells were immunoblotted for pAkt or total Akt under reducing conditions. (C) Serum-starved LECs were treated with Polydom (1 μg/ml) in medium with or without 0.5% FBS at 37°C for 30 min, followed by immunostaining for VE-cadherin (green) and FOXO1 (red). Nuclei were stained with Hoechst 33342 (blue). Cells in which FOXO1 was excluded from the nucleus (arrows) were frequently observed in the presence of Polydom, while FOXO1 was detected exclusively in the nucleus (closed arrowheads) or both the nucleus and cytoplasm with reduced signal intensity (open arrowheads) in the absence of Polydom. Bar, 20 μm. (D) Quantification of the percentages of cells showing FOXO1 localization in the nucleus only (black), in both the nucleus and the cytoplasm (gray), and in the cytoplasm only (white). The data represent means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 (n = 3). (E) Whole-mount immunofluorescence staining of the skin of E17.5 wild-type (Polydom+/+, upper panels) and Polydom-deficient mice (Polydom−/−, lower panels) for VEGFR3 (green, used as a lymphatic vessel marker) and Foxo1 (red). Nuclei were stained with Hoechst 33342 (blue). Magnified views of the boxed areas are shown below. Individual nuclei are encircled with dotted lines in the magnified views for Foxo1 staining. Bars, 20 μm. (F) Quantification of the percentages of VEGFR3-positive cells showing Foxo1 localization in the nucleus only (black), both in the nucleus and the cytoplasm (gray), and in the cytoplasm only (white) in Polydom+/+ and Polydom−/− mice. The data represent means ± SD. ***P < 0.001 (n = 3 per genotype). Source data are available for this figure: SourceData F4.