LMP1 decreases p27 expression and increases B-cell numbers in the pretumor spleens of λ-MYC mice. (A) Spleens were isolated from 4-week-old mice at the pretumor stage, and spleen weight as a percentage of body weight is shown. Lymphocytes were isolated from spleens of pretumor mice, and their B-cell numbers (B) and percentages (C) were calculated by B220 positivity using flow cytometry. (D) The percentage of IgM-positive cells to total splenic B cells was calculated by staining with anti-IgM. (E) The cell cycle phases of purified splenic pretumor B cells from 4-week-old mice are shown. Purified pretumor B cells were fixed in 70% ethanol and stained with a Ki-67–FITC antibody. DNA was stained with propidium–iodide/ribonuclease staining buffer according to the manufacturer’s instructions (BD Biosciences). (F) Representative immunohistochemical analysis of 4-week-old mouse spleens from each genotype stained with B220 (original magnification ×4). Splenomegaly was observed in LMP2A/λ-MYC and LMP1/LMP2A/λ-MYC spleens. (G) Representative immunoblots of p27kip1 expression in purified splenic B cells from 4-week-old mice. (H) P27kip1 expressions were normalized to Gapdh (a loading control). Dot plot graphs represent the mean ± standard deviation. P values were calculated by unpaired t test, *P < .05, **P < .01, ***P < .001, and ****P < .0001.