p16INK4a reactivation limits growth of ALCL cell lines. (A) Western blotting analysis of pRb, p16INK4a, phosphorylated pRb (serine 807-811), NPM-ALK, and tubulin expression in 6 different ALCL cell lines (as indicated). (B) Left panel: Western blot analysis of NPM-ALK, p16INK4a, and tubulin expression at different times (as indicated) in SU-DHL-1 cells treated with doxocyclin to induce shRNA interference of NPM-ALK. Right panel: p16INK4a mRNA quantification by qPCR analysis with specific primers. Data were standardized with ribosomal RNA and normalized against control untreated cells. (C) Karpas299 and DEL cell lines were infected with control (Ctrl) and p16INK4a-expressing retroviruses. Left panel: at day 4, cells were collected and expression of p16INK4a, phosphoRb, and tubulin evaluated by Western blot. Right panels: infected cells were selected, plated, and counted daily. (D) Karpas299 and DEL cell lines were treated with 5-azacytidine (AZA) for 6 days. Left panel: at day 5, cells were collected and expression of p16INK4a, phosphoRb, and tubulin evaluated by Western blot. Right panels: cells were plated and counted daily.