Figure 3.
NAMPT/SIRT2-mediated LMO2 deacetylation is essential for LMO2 interaction with LDB1, activation of TAL-1 complex, and proliferation of T-ALL cells. (A) Co-IP of LMO2 and LDB1 from lysates of MOLT-4 cells treated with 10 nM FK866 for 72 hours. IP and immunoblotting were performed using the indicated antibodies. Representative images are shown. Bar graphs of the LDB1/LMO2 binding ratio indicate the LDB1 protein signal divided by the LMO2 protein signal in IP samples. Data show means ± SD from 3 independent experiments (*P < .05). (B) Duolink in situ PLAs of MOLT-4 cells treated with 10 nM FK866 or 100 nM AC93253 for 48 hours. Cells were stained with anti-LMO2 and anti-LDB1 antibodies. Scale bars: 10 μm. (C) WT or SIRT2-knockout (SIRT2KO) HEK293FT cells were transfected with a GYPA reporter and the indicated expression constructs. Transfected cells were cultured for 24 hours; activation of the GYPA reporter was measured as described in "Methods." Signals measured in cells cotransfected with LMO2 and GYPA constructs were normalized to those in cells without an LMO2 expression plasmid (defined as 1). Data show means ± SD (n = 8; *P < .05). (D) mRNA expression levels of the indicated genes in MOLT-4 cells treated with 10 nM FK866, 100 nM AC93253, or DMSO for 72 hours were assessed using quantitative RT-PCR. Target gene/GAPDH mRNA expression ratios are shown. Data, expressed as arbitrary units (AUs), are means ± SD from 3 independent experiments, each in triplicate (*P < .05 compared with DMSO-treated cells). (E-G) Xenotransplantation of T-ALL cells into zebrafish embryos. Zebrafish embryos were transplanted with labeled primary T-ALL cells (E), MOLT-4 cells transduced with a WT (F), or K74/78R mutant LMO2-expressing lentivirus construct (G). FACS, fluorescence-activated cell sorting. Xenotransplanted embryos were treated with 800 nM FK866 or DMSO for 2 days. Numbers of transplanted cells were assessed as described in "Methods." Data represent percentage of labeled cells normalized to the average percentage of labeled cells in the DMSO-treated group. The black lines represent mean ± SD. Each dot represents 4 embryos pooled as 1 biological sample. The number of analyzed embryos is indicated in the lower panel of each figure (***P < .0001; **P < .001). (H) Representative western blot images of cell lysates of MOLT-4 cells transduced with the indicated lentivirus constructs using anti-LMO2 and anti-β-actin antibodies. NS, not significant.

NAMPT/SIRT2-mediated LMO2 deacetylation is essential for LMO2 interaction with LDB1, activation of TAL-1 complex, and proliferation of T-ALL cells. (A) Co-IP of LMO2 and LDB1 from lysates of MOLT-4 cells treated with 10 nM FK866 for 72 hours. IP and immunoblotting were performed using the indicated antibodies. Representative images are shown. Bar graphs of the LDB1/LMO2 binding ratio indicate the LDB1 protein signal divided by the LMO2 protein signal in IP samples. Data show means ± SD from 3 independent experiments (*P < .05). (B) Duolink in situ PLAs of MOLT-4 cells treated with 10 nM FK866 or 100 nM AC93253 for 48 hours. Cells were stained with anti-LMO2 and anti-LDB1 antibodies. Scale bars: 10 μm. (C) WT or SIRT2-knockout (SIRT2KO) HEK293FT cells were transfected with a GYPA reporter and the indicated expression constructs. Transfected cells were cultured for 24 hours; activation of the GYPA reporter was measured as described in "Methods." Signals measured in cells cotransfected with LMO2 and GYPA constructs were normalized to those in cells without an LMO2 expression plasmid (defined as 1). Data show means ± SD (n = 8; *P < .05). (D) mRNA expression levels of the indicated genes in MOLT-4 cells treated with 10 nM FK866, 100 nM AC93253, or DMSO for 72 hours were assessed using quantitative RT-PCR. Target gene/GAPDH mRNA expression ratios are shown. Data, expressed as arbitrary units (AUs), are means ± SD from 3 independent experiments, each in triplicate (*P < .05 compared with DMSO-treated cells). (E-G) Xenotransplantation of T-ALL cells into zebrafish embryos. Zebrafish embryos were transplanted with labeled primary T-ALL cells (E), MOLT-4 cells transduced with a WT (F), or K74/78R mutant LMO2-expressing lentivirus construct (G). FACS, fluorescence-activated cell sorting. Xenotransplanted embryos were treated with 800 nM FK866 or DMSO for 2 days. Numbers of transplanted cells were assessed as described in "Methods." Data represent percentage of labeled cells normalized to the average percentage of labeled cells in the DMSO-treated group. The black lines represent mean ± SD. Each dot represents 4 embryos pooled as 1 biological sample. The number of analyzed embryos is indicated in the lower panel of each figure (***P < .0001; **P < .001). (H) Representative western blot images of cell lysates of MOLT-4 cells transduced with the indicated lentivirus constructs using anti-LMO2 and anti-β-actin antibodies. NS, not significant.

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