Fig. 4.

Pharmacologic depletion of PI(3)P blocks ZFYVE21 expression. Human umbilical vein endothelial cells (HUVECs) transfected with control or ZFYVE21 siRNA were treated with panel reactive antibody (PRA) for 4 h prior to quantitative reverse transcriptase PCR (qRT-PCR) (a) or prior to EC:T cell coculture for 7 days (b). *p < 0.05, analysis of variance. Lipid reporter mean fluorescent intensity (MFI) of PI(3)P (2XFYVE-GFP) on C9+Rab5+ vesicles following drug pretreatment with SAR405 (5 nM), PIKIII (5 nM), and KU-55933 (10 μM) for 30 min prior to addition of PRA sera for 30 min (c). Western blotting of HUVECs pretreated with inhibitors for 30 min prior to addition of PRA for 30 min (d). qRT-PCR analysis of HUVECs treated with PRA sera for 4 h following drug pretreatment (e). **p < 0.001. EC:T cell cocultures were performed using HUVECs pretreated with drug(s), and the percentage of proliferating memory T cells were calculated (f). Lipid reporter MFI of PI(3)P (2XFYVE-GFP) on C9+Rab5+ vesicles following drug pretreatment with miltefosine (25 μM) for 30 min prior to addition of PRA sera for 30 min (g). Western blotting of HUVECs pretreated with miltefosine or AktIII for 30 min prior to addition of PRA for 30 min (h). HUVECs stably transduced with nuclear factor (NF)-κB luciferase reporter were treated with varying doses of miltefosine prior to addition of PRA (25% v/v in gelatin veronal buffer) or tumor necrosis factor-α (10 ng/mL) for 6 h (i). qRT-PCR of HUVECs following PRA sera treatment for 4 h with miltefosine (25 μM, j). EC:T cell cocultures using HUVECs pretreated with miltefosine prior to addition of CFSE-labeled CD4+CD45RO+ T cells (k). CD4+CD45RO+ T cells were injected over confluent PRA-treated HUVEC monolayers and adherent cells were quantified (l). qRT-PCR analyses were performed using technical triplicates, and each experiment was repeated 2–4 times. EC:T cell cocultures used 6 technical replicates for each group and each experiment was repeated 2–3 times. Lipid reporter analyses used 4–6 technical replicates and each experiment was repeated 3 times. Western blot analyses were repeated two to five times. NF-κB luciferase assays were performed using technical triplicates, and each experiment was repeated three times. Flow chamber studies were performed using technical triplicates with quantifications of ≥3 high-powered fields per sample, and each experiment was performed twice. Representative data shown **p < 0.001