Figure 2.
The NAMPT/SIRT2 pathway activates LMO2 by deacetylation in T-ALL cells. (A) Schematic of the NAMPT-NAD+-SIRTs pathway. (B) Reporter gene assay using GYPA reporter construct. HEK293T cells were transfected with a GYPA reporter and the indicated expression constructs. Transfected cells were cultured in the presence or absence of FK866 for 24 hours, and the activation of the GYPA reporter was measured as described in “Methods." The same concentration of DMSO was added as a vehicle control. Data show means ± SD (n = 8). *P < .05. (C) Live-cell counting of MOLT-4 cells treated with 10 nM FK866 or 100 nM AC93253. The same concentration of DMSO was added as a vehicle control. Dead cells were excluded by trypan blue dye staining. Data represent mean ± SD from 3 independent experiments, each in triplicate (*P < .05 compared with DMSO-treated cells). (D) Live-cell counting of primary blasts from T-ALL patients (n = 3) treated with 10 nM FK866 or 100 nM AC93253 for 48 or 72 hours. The same concentration of DMSO was added as a vehicle control. Dead cells were excluded by trypan blue dye staining. Live-cell numbers are normalized to those obtained in samples treated with DMSO (defined as 1). Data represent means ± SD of triplicate determinations (*P < .05 compared with DMSO-treated cells). Duolink in situ proximity ligation assay (PLA) of MOLT-4 cells (E) or primary blasts (F) from T-ALL patient (patient no. 3) treated with 10 nM FK866 or 100 nM AC93253 for 48 hours using anti-LMO2 and anti-acetylated lysine antibodies. The same concentration of DMSO was added as a vehicle control. Representative images are shown. Scale bars: 10 μm. (G-H) Proliferation of MOCK, WT-LMO2, or K74/78R LMO2-expressing MOLT4 cells (2 × 104 cells/well of a 96-well plate) was evaluated over time with the IncuCyte S3 Live-Cell Analysis System. The experiment was performed twice, each in triplicate. (I) Co-IP assay of SIRT2 and LMO2 protein interaction in lysates of HEK293T cells transfected with SIRT2 and LMO2 expression vectors. IP and immunoblotting were performed using the indicated antibodies. Representative images are shown. (J) Duolink in situ PLAs in MOLT-4 cells and primary blasts of T-ALL patients. Cells were stained with anti-LMO2 and anti-SIRT2 antibodies. Representative images are shown. Scale bars: 10 μm. RLU, relative light unit.

The NAMPT/SIRT2 pathway activates LMO2 by deacetylation in T-ALL cells. (A) Schematic of the NAMPT-NAD+-SIRTs pathway. (B) Reporter gene assay using GYPA reporter construct. HEK293T cells were transfected with a GYPA reporter and the indicated expression constructs. Transfected cells were cultured in the presence or absence of FK866 for 24 hours, and the activation of the GYPA reporter was measured as described in “Methods." The same concentration of DMSO was added as a vehicle control. Data show means ± SD (n = 8). *P < .05. (C) Live-cell counting of MOLT-4 cells treated with 10 nM FK866 or 100 nM AC93253. The same concentration of DMSO was added as a vehicle control. Dead cells were excluded by trypan blue dye staining. Data represent mean ± SD from 3 independent experiments, each in triplicate (*P < .05 compared with DMSO-treated cells). (D) Live-cell counting of primary blasts from T-ALL patients (n = 3) treated with 10 nM FK866 or 100 nM AC93253 for 48 or 72 hours. The same concentration of DMSO was added as a vehicle control. Dead cells were excluded by trypan blue dye staining. Live-cell numbers are normalized to those obtained in samples treated with DMSO (defined as 1). Data represent means ± SD of triplicate determinations (*P < .05 compared with DMSO-treated cells). Duolink in situ proximity ligation assay (PLA) of MOLT-4 cells (E) or primary blasts (F) from T-ALL patient (patient no. 3) treated with 10 nM FK866 or 100 nM AC93253 for 48 hours using anti-LMO2 and anti-acetylated lysine antibodies. The same concentration of DMSO was added as a vehicle control. Representative images are shown. Scale bars: 10 μm. (G-H) Proliferation of MOCK, WT-LMO2, or K74/78R LMO2-expressing MOLT4 cells (2 × 104 cells/well of a 96-well plate) was evaluated over time with the IncuCyte S3 Live-Cell Analysis System. The experiment was performed twice, each in triplicate. (I) Co-IP assay of SIRT2 and LMO2 protein interaction in lysates of HEK293T cells transfected with SIRT2 and LMO2 expression vectors. IP and immunoblotting were performed using the indicated antibodies. Representative images are shown. (J) Duolink in situ PLAs in MOLT-4 cells and primary blasts of T-ALL patients. Cells were stained with anti-LMO2 and anti-SIRT2 antibodies. Representative images are shown. Scale bars: 10 μm. RLU, relative light unit.

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