KLF2 and KLF4 regulate expression of endothelial TM. (A) Human umbilical vein endothelial cells (HUVECs) were transduced with lentivirus encoding shKRIT1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus control encoding shControl (for shKRIT1) or GFP-Control (for KLF2 or KLF4) was measured by RT-qPCR (SEM, n = 5).³⁵  (B) Analysis of THBD mRNA levels by RT-qPCR in HUVECs transduced with lentivirus encoding KLF2 or KLF4 compared with lentivirus encoding GFP as control (SEM, n = 3 or 4). (C) Analysis of TM protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4, as assessed by western blot analysis; lentivirus encoding GFP was used as control (SEM, n = 3). (D) Relative abundance of TM in GFP⁺ HUVECs transduced with lentivirus encoding KLF4-GFP or GFP as control, as assessed by flow cytometry. (E) Mean fluorescent intensity (MFI) of TM in KLF4-GFP+ cells compared with control GFP⁺ transduced HUVEC cells in (D) (SEM, n = 3). (F) Analysis of KLF2 mRNA levels by RT-qPCR in hCMEC/D3 cells transduced with lentivirus encoding shKRIT1 or scrambled control, followed by transfection with KLF2 and KLF4–specific small interfering RNAs (siRNAs; siK2/K4) or small interfering RNA control (siCtrl) (SEM, n = 4). (G) Analysis of KLF4 mRNA levels by RT-qPCR in hCMEC/D3 cells transduced with lentivirus encoding shKRIT1 or lentivirus encoding scrambled control, followed by transfection with KLF2 and KLF4–specific siRNAs (siK2/K4) or siRNA control (siCtrl) (SEM, n = 4). (H) Silencing of KLF2 and KLF4 (siK2/K4) using specific siRNAs prevented increased TM expression in KRIT1-depleted hCMEC/D3 cells. Cells were transduced with lentivirus encoding shKRIT1 or with lentivirus encoding shControl (SEM, n = 4). *P < .05, *P < .01, ***P < .001, Student t test.