American Society of Hematology

Figure 5

$Figure 5. Endothelial invasion of fibrin matrix in vitro is decreased by PI3Kβ loss and is rescued by PTEN inactivation. Knockdown of p110β in human ECs was performed as in supplemental Figure 3. Where indicated, nontransfected (NT) ECs or cells transfected with p110β (Siβ) or nonspecific siRNA (NS) were labeled with CellTracker green, cultured on Cytodex beads, embedded in a 3D fibrin gel in EGM2 for 18 hours, then imaged by fluorescence microscopy (A). The number of invading EC tip cells (B left panel) and distance of sprout extension (B, right panel) into the gel among p110β-knockdown vs control ECs is quantitated (mean ± SEM; n > 5 independent experiments; *P < .05, Siβ vs NS). (C) ECs were pretreated with TGX-221 (100 nM) or vehicle for 60 minutes, then evaluated for sprouting (left panel) and length of sprout extension (right panel; *P < .05 vs control). Siβ- or NS-transfected ECs were labeled with CellTracker red or green respectively, then equal numbers were mixed and cultured on Cytodex beads for suspension in fibrin gels. The fraction of sprouts with a leading Siβ or NS EC was quantitated (D; n = 3 independent experiments; *P < .05, Siβ vs NS). (E) ECs were transfected with siRNAs against p110β and/or PTEN, then evaluated for sprout formation (n = 3 independent experiments; * P < .05 vs the comparator group as indicated).$

Endothelial invasion of fibrin matrix in vitro is decreased by PI3Kβ loss and is rescued by PTEN inactivation. Knockdown of p110β in human ECs was performed as in supplemental Figure 3. Where indicated, nontransfected (NT) ECs or cells transfected with p110β (Siβ) or nonspecific siRNA (NS) were labeled with CellTracker green, cultured on Cytodex beads, embedded in a 3D fibrin gel in EGM2 for 18 hours, then imaged by fluorescence microscopy (A). The number of invading EC tip cells (B left panel) and distance of sprout extension (B, right panel) into the gel among p110β-knockdown vs control ECs is quantitated (mean ± SEM; n > 5 independent experiments; *P < .05, Siβ vs NS). (C) ECs were pretreated with TGX-221 (100 nM) or vehicle for 60 minutes, then evaluated for sprouting (left panel) and length of sprout extension (right panel; *P < .05 vs control). Siβ- or NS-transfected ECs were labeled with CellTracker red or green respectively, then equal numbers were mixed and cultured on Cytodex beads for suspension in fibrin gels. The fraction of sprouts with a leading Siβ or NS EC was quantitated (D; n = 3 independent experiments; *P < .05, Siβ vs NS). (E) ECs were transfected with siRNAs against p110β and/or PTEN, then evaluated for sprout formation (n = 3 independent experiments; * P < .05 vs the comparator group as indicated).

This Feature Is Available To Subscribers Only