NFATc1 regulates c-myc protein expression in DLBCL. (A) DLBCL MS, SUDHL4 and OCI-LY10 cells were treated with FK-506 (2.5 and 5 μg/mL) for 24 hours. Nuclear extracts (25 μg) were subjected to Western blotting for NFATc1, c-myc, and Oct-1 (loading control). (B) Purified RNA from control and FK-506 treated cells from part A was analyzed for c-myc mRNA expression. (C) DLBCL MS cells were transfected with a control shRNA (NC) or NFATc1 shRNA no. 1-4. Forty-eight hours after transfection, cells were harvested and nuclear extracts were purified. Nuclear extracts (25 μg) were subjected to Western blotting for NFATc1, c-myc, and Oct-1 (loading control). Densitometry analysis was performed for NFATc1 and c-myc protein bands (bottom graph). (D) DLBCL MS, SUDHL4, and OCI-LY10 cells were transfected with a control shRNA or an NFATc1 no. 2 shRNA. Forty-eight hours after transfection, purified RNA was analyzed for NFATc1 and c-myc mRNA expressions. (E) Exogenous expression of NFATc1 induces c-myc protein expression. DLBCL DS cells were infected with a retroviral vector containing a mutant NFATc1 (caNFATc1-GFP). After 48 hours of incubation, nuclear extracts (25 μg) were purified and analyzed for expression of NFATc1, c-myc, and Oct-1 proteins by Western blotting. (F) NFATc1-GFP fusion protein expression in infected DS cells was analyzed by confocal microscopic analysis, which indicates punctate nuclear and cytoplasmic expression of dephosphorylated NFATc1.