Table 1.
Mutation analysis of LM-derived cell-lines.
Fig 1.
Culture of lymphatic endothelial cells and fibroblasts.
Immunocytology showing CD31 (green) and PROX1 (magenta). Nuclei are counter-stained with DAPI (blue). A) Foreskin-derived HD-LECc4; B) Foreskin-derived HD-LECc2; C) Patient-derived Ly-LEC-12, D) Patient-derived Ly-LEC-2; E) Patient-derived Ly-LEC-10; F) Patient-derived fibroblasts (Ly-F-12) do not express CD31 or PROX1. Bar = 45 μm in A-D,F, and 90 μm in E.
Fig 2.
Patient-derived fibroblasts (Ly-F-12).
A) Phase-contrast image showing typical morphology. B) Anti-αSMA staining. C) Anti-vimentin staining. D) Negative control without primary antibodies. Note that all fibroblasts express αSMA and vimentin. Bar = 50μm.
Fig 3.
A,B. Phospho-AKT-Ser473 Western blot analysis of lymphatic endothelial cells.
Note that phospho-AKT-Ser473 (pAKT) is highly expressed in lymphatic malformation-derived LECs (Ly-LEC1, 2, 10, and 12), but not in normal HD-LECc3 and c4. Total AKT (tAKT) is found in all cell lines. Antibodies against α-tubulin were used as loading control.
Fig 4.
Phospho-ERK Western blot analysis of lymphatic endothelial cells.
Note that phospho-ERK (pERK) is highly expressed in lymphatic malformation-derived Ly-LECs (Ly-LEC1, 10, and 12), but not in normal HD-LECc3 and c4. Ly-LEC2, which contain a Glu109del in PIK3CA, are similar to normal LECs, which contain clear levels of total ERK (tERK). Antibodies against α-tubulin were used as loading control.
Fig 5.
Proliferation studies with PIK3CA inhibitor Buparlisib (BKM120) on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.
Fig 6.
Proliferation studies with PIK3CA inhibitor Wortmannin on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.
Fig 7.
Proliferation studies with PIK3CA inhibitor Ly294002 on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.
Fig 8.
Proliferation studies with PIK3CD inhibitor CAL-101 (Idealisib) on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.
Fig 9.
Proliferation studies with AKT inhibitor MK2206 on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.
Fig 10.
Proliferation studies with multi-kinase inhibitor Sorafenib on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.
Fig 11.
Proliferation studies with mTOR inhibitor rapamycin on Ly-LEC (left side, A—C) and HD-LEC (right side, D—F), after 0, 24, 48 and 72 hours (h).
0 h values were set to 100%. Concentrations are indicated. Shown are the mean values of n = 3 independent experiments with each 5–8 replicates.
Table 2.
Primers used for mutation analyses of PIK3CA.