Fig. 3. DEK overexpression stimulates transformation in vivo in cooperation with the HPV oncoproteins and oncogenic ras.

A, B. Nude mouse injections. 5× 10⁶ NIKs transduced with hRas, either E7 or E6/E7 and either empty R780 or R780-DEK vector were injected into the flanks of athymic nude mice. The R780 controls were injected into the left flank while DEK overexpressing cells were injected on the right flank of each mouse. Tumor formation (A) as well as tumor volume (B) were monitored over two months. Tumor volume was calculated using the following formula: length × width² × π/6. One asterisk represents a p-value of < 0.05 while two asterisks represent a p-value of < 0.01. Experiments containing 3-4 mice per group were performed three independent times. C. Tumor morphology in the presence and absence of overexpressed DEK. Tumors from (A) were fixed, embedded in paraffin and sectioned. Sections were then stained with hematoxylin/eosin and pictures were taken at 50 and 200 fold magnification for the top and bottom panels, respectively. D. Immunohistochemistry for phosphorylated (Ser10) Histone H3 was performed on paraffin sections that were baked, deparaffinized, rehydrated and subjected to antigen retrieval. Sections were blocked using goat antiserum in phosphate buffered saline. Primary phosphorylated (Ser10) Histone H3 1:1000 (US Biological) antibodies were diluted in blocking solution, applied to tissue sections and incubated overnight at 4°C Antibody staining was detected with Vectastain Elite ABC and DAB Substrate Kits (Vector Laboratories, Inc.). Counts represent evaluation of 300 tumor cells representing 2-5 tumor sections/mouse and 3 mice per group. The asterisk represents a p value of less than 0.05, and pictures were taken at 1000 fold magnification.