E2F3 contributes to lymphomagenesis of PBL. (A) Relative mRNA expression of E2F3 and target genes involved in cell cycle, in PBL-1 E2F3^mut1 and E2F3^mut2 (green) normalized to WT cells (gray). (B) Representative western blot images of E2F3 and its targets involved in cell cycle, in PBL-1 WT, E2F3^mut1, and E2F3^mut2 cells. (C) The relative protein expression normalized to PBL-1 cells. (D) Cell cycle was assessed by flow cytometry through TO-PRO-3 (APC) staining (left), and the percentage of cells in each cell cycle phases are shown in PBL-1 WT, E2F3^mut1, and E2F3^mut2 cells (right). (E) Migration and invasion of WT, E2F3^mut1, and E2F3^mut2 cells were determined at 48 hours in 5-μm-pore membranes of transwell inserts. The cells that migrated and invaded the lower chamber were fixed, stained with Giemsa, and counted (left). Relative migration and invasion plots (%) are shown (right). (F) Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and relative proliferation of E2F3 mutant cells normalized to WT ones is shown. (G) PBL-1 cells were inoculated in the chicken CAM in vivo model. The number of tumoral cells in the tumors developed after 7 days after cell inoculation was assessed by CD138-FITC staining. Flow cytometry plot of cells expressing CD138 (left) and the relative CD138⁺ cells in E2F3^mut tumors normalized to WT ones (right). (H) Representative images of hematoxilin-eosin (HE), MUM-1, and Ki67 immunohistochemistry staining from formalin-fixed, paraffin-embedded sections of CAM samples (left); and relative proliferation of WT and E2F3^mut tumors (right). All in vitro experiments were performed in biological triplicates and technical duplicates unless otherwise specified; in vivo CAM experiments were performed in 5 to 10 eggs. Statistical significance was determined by t test or analysis of variance (ANOVA) test or t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. FITC, fluorescein; HE, hematoxylin and eosin.