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P. Lin et al.
shRNA-1 and Dusp1 shRNA-1 were chosen because the data indicated that both of them mediated the best knock- down efficiency in human and mouse MDS cell lines, respectively (Online Supplementary Figure S6A-D). Downregulation of DUSP1 by lentivirus shRNA could sensitize SKM-1 cells to Ara-C treatment (72 h Ara-C IC50, scramble shRNA 1.91 mM vs. DUSP1 shRNA-1 1.23 mM; P=0.002) (Figure 5B). Treatment of the NRF2 agonist SFN significantly mitigated Ara-C toxicity in scramble shRNA SKM-1 cells (72 h Ara-C IC50, control treatment 2.05 mM vs. SFN treatment 2.50 mM; P=0.044). Significantly, DUSP1 shRNA on NRF2 elevated SKM-1 cell lines abrogate Ara-C resistance (72 h Ara-C with SFN treatment IC50, scramble shRNA 2.50 mM vs. DUSP1 shRNA-1 1.35 mM; P=0.020) (Figure 5C). In MLLPTD/WT /RUNX1-S291fs cells, Dusp1 downregulation could sensi-
tize MDS mouse cells to Ara-C treatment (48 h Ara-C IC50, scramble shRNA 0.20 μM vs. Dusp1 shRNA-1 0.15 mM; P=0.001) (Online Supplementary Figure S6E). Consistent with the data in SKM-1 cells, Dusp1 shRNA could also re-sensitize Ara-C-resistant MDS mouse cells [48 h Ara-C IC50, scramble shRNA with control treatment 0.20 mM vs. scramble shRNA with SFN treatment 0.22 mM (P=0.025) scramble shRNA with SFN treatment 0.22 mM vs. Dusp1 shRNA-1 with SFN treatment 0.16 mM (P=0.001)] (Online Supplementary Figure S6F). Knockdown of DUSP1 led to an increase in the apoptotic rate of SKM- 1 treated with Ara-C compared to scramble shRNA SKM- 1 cells (Figure 5D). DUSP1 silenced SKM-1 cell lines with Ara-C treatment also tended to be arrested in the S phase (Figure 5E). These results indicated that DUSP1 was a downstream gene of NRF2 and partially responsible for
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Figure 6. Knockdown of NRF2 significantly sensitizes myelodysplastic syndrome (MDS) cells to cytarabine (Ara-C) in vivo. (A) Flow cytometry showed appearance of human CD45+ cells in liver tumors of scramble, NRF2 and DUSP1 shRNA SKM-1 transplanted NSGS mice. (B) Liver tumors in scramble, NRF2 and DUSP1 shRNA SKM-1 transplanted NSGS mice with phosphate buffer saline (PBS) or Ara-C treatment. (C) Liver tumor volumes were significantly smaller in NRF2 or DUSP1 shRNA SKM-1 transplanted MDS mice treated with Ara-C compared with PBS. *P<0.05; **P<0.01; ***P≤0.001. g: grams.
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haematologica | 2019; 104(3)