Lck synergizes with Bfl-1 in lymphomagenesis. (A) FL5.12 cells stably expressing constitutively active Lck Y505F alone or together with GFP-Bfl-1 or GFP-Bfl-1ΔC were analyzed by Western blot with anti-Lck, anti-GFP, or anti–β-actin. (B) Kaplan-Meier curves show accelerated tumorigenesis in NCR nude mice transplanted intravenously with FL5.12 cells expressing GFP-Bfl-1ΔC/Lck Y505F (green, n = 4) or GFP-Bfl-1/Lck Y505F (purple, n = 4) compared with those expressing Lck Y505F alone (blue, n = 4). (C top) Western blot showing that Lck shRNAs 1 and 2 reduce endogenous mLck levels in GFP-Bfl-1ΔC/p53DD tumor 3–derived cells at 4 days after nucleofection after sorting for high GFP expression (shRNAs 1a and 2, lanes 2-3) versus cells nucleofected with nonspecific scrambled shRNA control (NS) sorted for high GFP expression, cells nucleofected with shRNA1 sorted for low GFP expression (shRNA#1b) as control or untransfected cells (U; lanes 1, 4-5). (Bottom) Quantification of Lck knockdown efficiency relative to the NS shRNA control. (D) Stable knockdown of mLck in GFP-Bfl-1ΔC/p53DD tumor 3–derived cells selectively reduces phospho-Akt, -IKKβ, and -MEK activation and leads to activation of phospho-JNK versus controls as seen by Western blot with phospho-Akt, -IKKβ, -MEK1/2, -JNK, -p38, or -ERK1/2. (E) Kaplan-Meier curves demonstrate that stable knockdown of endogenous mLck with shRNA #1a (green, n = 4) significantly delays tumor formation in NCR nude mice injected intravenously with GFP-Bfl-1ΔC/p53DD-tumor 3–derived cells (ΔC/DD #3) versus those injected with tumor 3 cells expressing the nonspecific shRNA control (NS; blue, n = 4; P = .001) or untreated cells (red, n = 6; P = .001). (F) Model representing Bfl-1 ubiquitin-mediated regulation and its role in tumor suppression.