Fig. 4.

APOD expression is dependent on NOTCH3. HDFNs were transiently transfected with control small interfering (si)RNA (siControl) or NOTCH3 siRNA (siNOTCH3) and cocultured with HUVECs. Forty-eight hours later, the cocultured cells were separated by anti-PECAM1-conjugated Dynabeads and total RNA was collected for qPCR to detect APOD RNA expression (A). APOD protein expression was determined by ELISA (B). NOTCH3 intracellular domain (NICD3) was overexpressed in mural cells by lentiviral infection. A lentivirus expressing GFP alone was used as control. Following infection, cells were analyzed for APOD expression by qPCR (C) and ELISA (D). *P < 0.05. Western blot analysis to demonstrate efficient knockdown of NOTCH3 by siRNA (E) and NICD3 overexpression (F). Tubulin expression was used as control.