Class I PI3K is involved in FLS formation with PI(3)P being a key downstream lipid. (A) TIRF and confocal imaging of representative FLS regions containing labeled SNX9 (TIRF), TOCA-1 (TIRF), or actin (maximally projected confocal side or Z views) treated with DMSO or 100 µM LY294002. Both SNX9 and actin but not TOCA-1 are reduced by the inhibitor. (B) TIRF and confocal imaging of representative FLS regions containing labeled SNX9, the PI(3)P probe mCh-2xFYVE (TIRF), or actin (maximally projected confocal side or Z views) grown on membranes containing 10% PI(4,5)P₂ or 10% PI(4,5)P₂ plus 5% PI(3)P, 5% PI(3,4)P₂ or 5% PI(3,4,5)P₃ and treated with either DMSO or 2 µM wortmannin. SNX9, mCh-2xFYVE, and actin are reduced on wortmannin treatment. Inclusion of 5% PI(3)P shows mCh-FYVE labeling and SNX9 recruitment in the presence of wortmannin and 5% PI(3,4)P₂ or 5% PI(3,4,5)P₃ do not. (C and D) Quantification of data in B. Each datapoint represents an individual imaging region (n = 24 regions for each treatment from two independent experiments). Lines indicate mean ± SEM. Statistical significance was assessed by ordinary one-way ANOVA with Holm–Sidak’s multiple comparisons test (Table S2). Scale bars, 10 µm. n.s., not significant.