p18Ink4c loss promotes emergence of tumor from a premalignant senescent lesion. A) Representative pineal sections of Irbp-Cyclin D1, p18Ink4c-/- mice at the indicated ages, with Ki67 immunostaining. Arrowheads mark examples of Ki67-positive cells. B) Immunoflourescence for H3K9me3 and corresponding DAPI staining of Irbp-Cyclin D1, p18Ink4c -/- pineal sections at 3.5 months. Asterisk denotes the emerging tumor; dashed line demarcates tumor from the senescent pineal lesion. Arrows indicate examples of cells with SAHF (positive for H3K9me3). C) Dual immunoflourescence for BrdU (green) and H3K9me3 (red) in Irbp-Cyclin D1, Ink4c-/- pineal section at P49. Red arrows indicate cells with H3K9me3 foci, white arrows cells positive for BrdU. D) Western blotting for the indicated proteins in representative tumors from Irbp-Cyclin D1, p53-/- (D1p53n) and Irbp-Cyclin D1, p18Ink4c-/- (D1p18n) mice, compared to a P10 Irbp-Cyclin D1 pineal gland (D1). HA: HA tag of the Cyclin D1 transgene. E) Representative western blot for the indicated proteins in tumor cells from Irbp-Cyclin D1, p53 -/- (D1p53nT) and Irbp-Cyclin D1, p18Ink4c -/- (D1p18nT) mice, before and after treatment with ionizing radiation or etoposide, as indicated. F) Western blotting for the indicated proteins in Irbp-Cyclin D1, p53 -/- (D1p53null) pineal lysates at the indicated ages and in tumor lysates as indicated.