Figure 6.

Calcium induces spinous differentiation through canonical Notch/RBP-J signaling. (A–C) Calcium induces RBP-J reporter gene activity (A), and endogenous K1/K10 gene expression (B) by a Notch/RBP-J-dependent mechanism, as this effect is blocked by expression of a dominant-negative RBP-J. (C) Stimulation of spinous markers by NICD1 is completely blocked in the absence of RBP-J. RBP-J reporter assays and quantifications of K1 and K10 were performed on FACS-isolated MK cultured in low-calcium (differentiation-restricted) or high-calcium (differentiation-promoting) medium, as indicated. Where noted, cells were also transduced with IRES-GFP retroviral expression vectors encoding NICD, dominant-negative RBP-J (RBP-DN), or empty vector (Co, control). Where indicated, cells were also treated with DAPT (N-[N-{3,5-difluorophenacetyl}-lalanyl]-S-phenylglycine t-butyl ester) to inhibit Notch processing and NICD production (Geling et al. 2002).