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. Author manuscript; available in PMC: 2015 May 15.
Published in final edited form as: Oncogene. 2013 Jun 10;33(20):2665–2673. doi: 10.1038/onc.2013.208

Figure 3. RAS pathway signaling, cell proliferation, and DNA damage.

Figure 3

[A] Arf−/− adenocarcinomas (AC) displayed a higher proliferation index than tumors from Arf+/+ littermates, as shown by phosphorylated histone H3 staining (**** P < 0.0001; n = 119 adenoma and 220 AC fields counted from Arf+/+; n = 22 adenoma and 829 AC fields from Arf−/− mice). [B] Phosphorylated histone H2A.X staining illustrates a marked increase in DNA damage in tumors from Arf-deficient mice (**** P < 0.0001; *** P = 0.0001; n = 85 adenoma and 307 AC fields counted from Arf+/+; n = 31 adenoma and 788 AC fields from Arf−/− mice). In addition to H2A.X+ cells occurring more frequently per field, damage foci were also larger and more numerous per cell in Arf−/− mice compared to Arf+/+. Note that the paucity of adenomas in Arf−/− animals prevented inclusion of additional fields in the statistical analysis of phospho-H3 and phospho-H2A.X staining. [C] Nuclear cyclin D1 and phospho-ERK1/2 expression were strongly upregulated in adenomas (top) and adenocarcinomas (bottom) from individual Arf-deficient mice (lanes 4–6 and 10–12) compared to Arf+/+ mice (lanes 1–3 and 7–9). β-actin is provided as loading control. β-tubulin and lamin B1 are included as markers of cytoplasmic and nuclear compartments, respectively. [D] Relative intensities of nuclear versus cytoplasmic cyclin D1 bands from [C] compared between genotypes at each tumor stage. All samples were normalized to β-actin (* P = 0.0114 adenomas, * P = 0.0308 ACs). [E] Expression of Ccnd1 mRNA is equivalent in adenocarcinomas from Arf+/+ and Arf−/− animals. Gapdh was used as endogenous control. Data is plotted as fold change compared to mean Arf+/+ value (n = 6 tumors per genotype; P = 0.6600).