Figure 5.

CRISPR/Cas9 down-regulation of G3BP1 in aortic VSM cell cultures reduces nuclear NFATc4 protein accumulation and NFATc4 association with OPN and TNAP promoters. Aortic VSM were transduced with antibiotic-selectable Cas9-expressing lentivirus. Cas9-expression pooled clones were then transduced with lentivirus expressing a G3BP1 guide RNA (gRNA), and NFATc4 and G3BP1 protein levels were assessed by Western blot analysis (A and B) or cultures were cross-linked and processed for ChIP assay (C). A, nuclear NFATc4 levels were down-regulated by G3BP1-directed gRNA. B, quantitative image analysis of relative NFATc4 expression in A. C, ChIP assay demonstrated reduced NFATc4 association with OPN and TNAP promoters in G3BP1-depleted VSM cells.