Figure 4. Aortic smooth muscle cells (SMCs) undergo RIP3-dependent necroptosis in vitro.

(A) Aortic SMCs isolated from C57BL/6 mice were transfected with control siRNA or RIP3 siRNA. Cells were treated with DMSO (vehicle control), TNFα or TNFα plus zVAD for 24 hours. Cells were stained with PE Annexin V and 7-AAD and analyzed by flow cytometry. Necrotic cells were identified as PE Annexin V⁺/7-AAD⁺. The Western blot shown on the rightmost confirms the gene silence efficacy and specificity of RIP3 siRNA. Cell lysates were collected 36 hours post-transfection. **P<0.005. Data represent mean±SEM. n=3. (B) Rip3^-/- aortic SMCs were infected with the empty adenoviral vector (AdNull) or the recombinant adenoviral vector expressing RIP3 (AdRIP3) at indicated multiplicity of infection (M.O.I.) units. After 24 hours, cells were treated with DMSO (vehicle control) or TNFα plus zVAD for 24 hours. Cell necrosis was determined by flow cytometry following staining with PE Annexin V and 7-AAD. Necrotic cells were identified as PE Annexin V⁺/7-AAD⁺. AdRIP3-mediated RIP3 expression in Rip3^-/- SMCs was confirmed by Western blot. *P<0.05, ****P<0.0001. Data represent mean±SEM. n=3∼5.