Figure 9.

CRISPR/Cas9 down-regulation of RIG-I/Ddx58 reduces osteogenic differentiation of aortic VSM cells. Aortic VSM were transduced with antibiotic-selectable Cas9-expressing lentivirus. Cas9-expression pooled clones were then transduced with lentiviruses expressing RIG-I/Ddx58 gRNA, and RIG-I protein levels were assessed by Western blot analysis (A). In parallel, cultures were grown under mineralizing conditions, and osteogenic re-programming assessed by RT-qPCR (B) or alkaline phosphatase enzyme activity (C). Note that down-regulation of VSM RIG-I (A) is again associated with reductions in the osteogenic potential of aortic VSM (B and C). *, **, ***, p ≤ 0.05, ≤ 0.01, and ≤ 0.001 versus corresponding controls.