Figure 6.
The NAMPT-NAD+-SIRT2 pathway plays an essential role in early-stage hematopoietic differentiation of iPS cells via LMO2 deacetylation. (A) Schematic of the serum- and feeder-free monolayer human iPS cells hematopoietic differentiation system. (B-C) The percentage of KDR+CD34+ cells was evaluated in differentiated iPS cells on day 6 treated with FK866 (B) or AC93253 (C). The same concentration of DMSO was used as a vehicle control. Data show means ± SD and are derived from 3 independent experiments, each in triplicate. *P < .05. (D-E) The percentage of CD43+ cells was evaluated by flow cytometry (D), and colony-forming unit assays were performed in differentiated iPS cells on day 13 treated with 100 nM AC93253 during differentiation (E). The same concentration of DMSO was used as a vehicle control. Data are mean ± SD derived from 3 independent experiments, each in triplicate. *P < .05. (F) Representative histograms of the Duolink-FACS analysis on the acetylation of the indicated proteins in differentiated iPS cells on day 6. Blue, signal from total protein; red, signal from acetylated protein; lime green, negative control. (G) Duolink in situ PLA assay in the differentiated iPS cells on day 6 of culture. Cells were stained with anti-LMO2 and anti-SIRT2 antibodies. Representative images are depicted. Scale bars: 10 μm. (H) Representative histograms of the Duolink-FACS analysis on the acetylation of LMO2 protein in differentiated iPS cells on day 6 in the absence or presence of 100 nM AC93253. The same concentration of DMSO was added as a vehicle control. Blue, signal from total protein; red, signal from acetylated protein; lime green, negative control. Graph bars of the total to acetylated protein Duolink-FACS signal ratio indicates the mean fluorescence intensity (MFI) of acetylated LMO2 protein signal divided by the MFI of total LMO2 protein signal. Data are mean ± SD from 3 independent experiments. *P < .05. (I) Duolink in situ PLA assay in the differentiated iPS cells on day 6 of culture treated with 100 nM AC93253. The same concentration of DMSO was added as a vehicle control. Cells were stained with anti-LDB1 and anti-LMO2 antibodies. Representative images are depicted. Scale bars: 10 μm. (J) mRNA expression levels of the indicated genes in differentiated iPS cells on day 6 of culture in the presence of 3 nM FK866 or 100 nM AC93253 were assessed using quantitative RT-PCR and expressed as AUs. The same concentration of DMSO was added as a vehicle control. Target gene ACTB mRNA expression ratio measured in the control DMSO-treated sample was normalized to 1. Data are mean ± SD from 3 independent experiments, each in triplicate. *P < .05 compared with DMSO-treated cells. ND, not detected. 

The NAMPT-NAD+-SIRT2 pathway plays an essential role in early-stage hematopoietic differentiation of iPS cells via LMO2 deacetylation. (A) Schematic of the serum- and feeder-free monolayer human iPS cells hematopoietic differentiation system. (B-C) The percentage of KDR+CD34+ cells was evaluated in differentiated iPS cells on day 6 treated with FK866 (B) or AC93253 (C). The same concentration of DMSO was used as a vehicle control. Data show means ± SD and are derived from 3 independent experiments, each in triplicate. *P < .05. (D-E) The percentage of CD43+ cells was evaluated by flow cytometry (D), and colony-forming unit assays were performed in differentiated iPS cells on day 13 treated with 100 nM AC93253 during differentiation (E). The same concentration of DMSO was used as a vehicle control. Data are mean ± SD derived from 3 independent experiments, each in triplicate. *P < .05. (F) Representative histograms of the Duolink-FACS analysis on the acetylation of the indicated proteins in differentiated iPS cells on day 6. Blue, signal from total protein; red, signal from acetylated protein; lime green, negative control. (G) Duolink in situ PLA assay in the differentiated iPS cells on day 6 of culture. Cells were stained with anti-LMO2 and anti-SIRT2 antibodies. Representative images are depicted. Scale bars: 10 μm. (H) Representative histograms of the Duolink-FACS analysis on the acetylation of LMO2 protein in differentiated iPS cells on day 6 in the absence or presence of 100 nM AC93253. The same concentration of DMSO was added as a vehicle control. Blue, signal from total protein; red, signal from acetylated protein; lime green, negative control. Graph bars of the total to acetylated protein Duolink-FACS signal ratio indicates the mean fluorescence intensity (MFI) of acetylated LMO2 protein signal divided by the MFI of total LMO2 protein signal. Data are mean ± SD from 3 independent experiments. *P < .05. (I) Duolink in situ PLA assay in the differentiated iPS cells on day 6 of culture treated with 100 nM AC93253. The same concentration of DMSO was added as a vehicle control. Cells were stained with anti-LDB1 and anti-LMO2 antibodies. Representative images are depicted. Scale bars: 10 μm. (J) mRNA expression levels of the indicated genes in differentiated iPS cells on day 6 of culture in the presence of 3 nM FK866 or 100 nM AC93253 were assessed using quantitative RT-PCR and expressed as AUs. The same concentration of DMSO was added as a vehicle control. Target gene ACTB mRNA expression ratio measured in the control DMSO-treated sample was normalized to 1. Data are mean ± SD from 3 independent experiments, each in triplicate. *P < .05 compared with DMSO-treated cells. ND, not detected. 

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