Figure 4. A 441bp DNA sequence within the 1.2kb fragemtn can mediate the regulation of ΔNp63 expression by inactivated FANCD2.

(A) The 1.2 kb DNA fragment was divided into three DNA segments with a size of 377, 401, or 441 bps. (B) 441 bp DNA fragment, but not the 377 and 403 bp ones (Supplementary Figure 2, left panel), can be pulled down more by FANCD2 antibodies in U2OS and HCT116 stably-transfected cells carrying an inactivated FANCD2 compared to corresponding control cells with an intact FA pathway. (C) the 441 bp DNA fragment can also be pulled down more by Flag antibodies in cells transiently co-transfected with Flag-mtFANCD2, but not Flag-wtFANCD2. A ChIP assay was performed in cells transiently transfected with pEGFP-Flag wtFANCD2 or mtFANCD2 by using Flag antibodies. ChIP-PCR primers were designed for three fragments shown in (A). The similar transfection efficiency was measured through western blotting by using antibodies against GFP or Flag (right panel). (D) K561R mtFANCD2 stimulates the 441-reporter activity. Similarly as previously done, three DNA segments were individually cloned into the upstream of pGL-3-promoter-reporter, and named the 377, 403, or 441 -reporters. 293T cells were transiently transfected respectively with these reporters and pEGFP-Flag plasmids containing wt or mtFNACD₂. All luciferase assays were normalized for transfection efficiency by renilla reporter activity. Western blot analysis with Flag or GFP antibodies was performed to verify expressions of both wt and mtFANCD2 proteins. The results shown are a representative of five independent experiments performed each time in triplicate, error bars indicating the standard deviation [the 377-reporter (not shown) produces luciferase activity similar to the one derived from the 403-reporter].