Fig. 8. Enhanced survival of Inv-Dsg2 keratinocytes in forced suspension culture is dependent on NF-B activation. (A) Clonal growth of control and Inv-Dsg2 cell lines after 0, 24, 48 and 72 hours in forced suspension culture was assessed by replating cells on tissue culture plastic and allowing cell proliferation for 7-9 days after replating. Inv-Dsg2 transgenic cells showed dramatically increased survival and re-growth compared to wild-type cells, in this setting. (B) Inv-Dsg2 transgenic cells were subjected to cell suspension culture for 48 hours with EGF and/or Bay11-7082, an IB-alpha phosphorylation inhibitor. Cell survival was assessed by replating cells on tissue cultured-treated plastic for 7 days. Activation of the EGF receptor with exogenous EGF further enhanced survival of Inv-Dsg2 transgenic cells in forced suspension cultures. Bay11-7082 inhibition of NF-B completely abolished cell survival in suspension culture. EGF-R activation was unable to counteract the effect of Bay11-7082. (C) Immunoblot analysis of NF-B p65 expression in wild-type and transgenic skin. Actin was used as a loading control. (D) Immunoblot results were confirmed by immunofluorescent staining, which showed the presence of NF-B p65 in nuclei of transgenic but not wild-type skin. Dotted lines demarcate dermal-epidermal junction. Bars, 50 µm.