ZFP521 regulates self-renewal ex vivo and facilitates MLL-AF9–mediated AML leukemia in mice. (A) Serial replating assays of HSCs transduced with lentiviruses encoding ZsGreen (control), ZFP521, or ZFP521(ΔNuRD). (B) Representative histograms (left) and quantitation (right) of DNA content per cell cycle from 3 independent experiments. The percentage of cells in S/G2/M phase of the cell cycle is presented as mean ± SEM. Unpaired t test: ***P < .001. (C) Wright-Giemsa staining (G, granulocyte; M, macrophage; PL, progenitor-like cell) of cultured HSC-derived cells ectopically expressing ZsGreen (control), ZFP521, or ZFP521(ΔNuRD). Scale bar represents 50 µm. (D) Heatmap showing relative expression (red [high expression] to blue [low expression]) of 26 conserved HSC-enriched genes, including the 6 indicated transcription factors within patient samples from 4 AML cytogenetic subtypes. See supplemental Table 4 for a complete gene list. (E-F) Kaplan-Meier survival curves following (E) primary and (F) secondary transplantation of MLL-AF9–transduced cells on a WT or Zfp521-deficient (KO) background, with the number (n) of mouse recipients indicated per group. Log-rank (Mantel-Cox) testing was conducted for statistical analyses; *P < .05, **P < .01. (G) Serial plating of bone marrow–derived MLL-AF9–transduced cells. Number of colonies at each passage is presented as mean ± SEM of 3 independent experiments. Unpaired t test: *P < .05, **P < .01. (H) Relative cell growth of MLL-AF9–positive (NOMO-1 and THP-1) or non–MLL-AF9 AML cells (OCI-AML3, HL-60, and U937) following doxycycline (+DOX)–induced shRNA-mediated ZNF521 knockdown (shZNF521) or scramble shRNA (shCONTROL) compared with the uninduced (−DOX) condition. Relative cell growth (+DOX/−DOX) values are presented as mean ± SEM of 3 independent experiments. Unpaired t test: *P < .05, **P < .01.