Fig. 1. IFN-γ stimulates RIG-I expression in human umbilical artery SMC in a concentration-dependent manner. (A) Human umbilical artery SMC were incubated with 0.01 ng/ml–25 ng/ml IFN-γ for 8 h, and total RNA was extracted. Single-strand cDNA was synthesized from 1 μg of total RNA, and RT-PCR analyses of mRNAs for RIG-I, α-actin and GAPDH were performed. (B) SMC were treated with IFN-γ for 24 h. Cells were lysed, and lysates were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane, and RIG-I protein with a molecular weight of 101 K was detected using rabbit anti-RIG-I antiserum and HRP-labeled anti-rabbit IgG.

Fig. 2. RIG-I is expressed in human umbilical artery SMC stimulated with IFN-γ in a time-dependent fashion. (A) Cells were stimulated with 10 ng/ml IFN-γ for up to 24 h, and RT-PCR analyses for RIG-I, IP-10, α-actin and GAPDH were performed. (B) After incubating with IFN-γ for up to 48 h, the cell lysate was subjected to western blot analysis for RIG-I.

Fig. 3. RIG-I protein is located in cytoplasm in human umbilical artery SMC. (A) SMC were stimulated with 10 ng/ml IFN-γ for 24 h, and nuclear and cytoplasmic fractions were prepared using a NE-PER reagent. Expression of RIG-I protein was examined by western blot analysis. (B) Immunofluorescent staining of RIG-I in human umbilical artery SMC. SMC were stimulated with 10 ng/ml IFN-γ for 24 h and subjected to immunofluorescent staining. Cells were fixed with 4% paraformaldehyde and incubated with an anti-RIG-I antibody or a control antibody, followed by the incubation with biotin-labeled anti-rabbit IgG and FITC-streptavidin. Cells were observed using a laser confocal microscope. Bar=10 μm.

Fig. 4. Immunohistochemical detection of RIG-I in vascular SMC in human tissues. SMC in tissues from three autopsied cases were stained using an anti-RIG-I antibody (A), an anti-α-actin antibody (B) or a control antibody (C). Nuclear counterstaining was performed. Case 1; abdominal aorta from 69 years old female whose death was attributed to pulmonary embolism after left hip replacement surgery. Case 2; abdominal aorta from 71 years old male died of gastric cancer. Case 3; abdominal aorta (3-1) and a vein in the abdominal aortic adventitia (3-2) from 18 years old female died of liver cirrhosis. Note that vascular SMC were stained positively in (A) and (B), but not in (C). Bar = 30 μm.