Fig. 3

Sec14l3 facilitates PLC-catalyzed PIP₂ hydrolysis induced by Wnt5b.

(A) PM isolation analysis of PM PIP₂ levels using PLCδ1-PH-GFP as probe in HEK293T cells. Quantification data from three independent experiments are shown as mean ± SEM (see also Figure 3—source data 1, *p<0.05). (B) Immunofluorescence of PLCδ1-PH-GFP in first panel (transfected with PLCδ1-PH-GFP) and endogenous PIP₂ in second panel shows PIP₂ accumulation in the PM of stable SEC14L2-knockdown HEK293T cells. Data are presented as mean ± SEM (see alsoFigure 3—source data 1, **p<0.01; n ≥ 50 cells from three separate experiments). Scale bar, 10 μm. (C) Immunofluorescence of PLCδ1-PH-mCherry (red, PIP₂ probe), β-catenin (green, PM marker) and DAPI (blue, nucleus marker) shows PIP₂ accumulation in the PM of Msec14l3 mutant cells. The first two whole embryo panels are 3D views of z-stacks (n = 30 for 4 hpf, n = 34 for 6 hpf), while the last panel is enlarged views of single z-stack pictures (z = 8 for 4 hpf, z = 7 for 6 hpf) from regions encompassed by white boxes. Scale bars, 100 μm for whole embryos; 25 μm for the enlarged columns. (D) Sec14l3 depletion compromises Wnt5b-induced degradation of PM PIP₂. Immunofluorescence of PLCδ1-PH-mCherry (red) and GFP (green, indicating Wnt5b-expressed cells) is shown. Mosaic expression of 100 pg wnt5b mRNA was created in embryos with even distribution of PLCδ1-PH-mCherry mRNA. White polygons outline GFP expressed cells and single z-stack pictures (z = 10) from numbered regions in the whole embryo panels (3D view of z-stacks) are enlarged. Scale bars, 100 μm for whole embryos; 25 μm for the enlarged panels. (E) sec14l3 overexpression inhibits accumulation of PIP₂ in wnt5b morphant embryos. Mosaic expression of sec14l3 by injecting 150 pg mRNA was created in embryos with even distribution of PLCδ1-PH-mCherry mRNA in std-MO or wnt5b-MO injected embryos. Single z-stack pictures (z = 11) from numbered regions in the whole embryo panels (3D view of z-stacks) are enlarged. (F) PM PIP₂ quantification of (C–E) by calculating intensity of (PM-Cytosol)/Cytosol PLCδ1-PH-mCherry. Data are shown as mean ± SEM. (see also Figure 3—source data 1, **p<0.01; *p<0.05; ns, non-significant; n ≥ 50 cells from 10 embryos in three independent experiments).