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. 2021 Dec 16;138(24):2526–2538. doi: 10.1182/blood.2021011711

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Figure 6. — TFAP4-mediated restriction of Erg is required for coupling of c-MYC–dependent proliferation and differentiation. (A) Scheme for in vivo assessment of expansion and differentiation of Tfap4^+/+, Tfap4^+/−, Tfap4^+/− Erg^+/−, and Erg^+/− CD19⁺ pro/pre-B cells transduced with a distinct retrovirus encoding mouse c-MYC (T58A). MSCV-based retroviruses expressing mouse MYC-ires-GFP and mouse MYC-ires-Thy1.1 were packaged by transiently transfecting PlatE cells with a helper plasmid, pCL-10A1. Viral supernatant harvested 48 hours after transfection was used to infect freshly isolated CD45.2 CD19⁺ BM cells from 6- to 14-week-old mice (age matched within the experimental groups) with the indicated genotypes at 1000g, 30°C for 1.5 to 2 hours in the presence of 10 mg/mL Polybrene. Transduced CD19⁺ BM cells from Tfap4^+/+ (WT) and Tfap4^+/− or Tfap4^+/− Erg^+/− or Erg^+/− mice were cotransferred into sublethally irradiated CD45.1 recipients at a 1:1 ratio. Comparable infection efficiency across genotypes when using MYC-GFP or MYC-Thy1.1 retroviruses was achieved, as shown in supplemental Figure 6H. Three weeks later, mice were euthanized, and the proportions of each transduced cell population and their differentiation status were analyzed. (B) Representative flow cytometry plots showing frequencies of Tfap4^+/+ (WT) and Tfap4^+/− (left panel), WT and Tfap4^+/− Erg^+/− (middle panel), and WT and Erg^+/− (right panel) donor-derived B220⁺ cells in the BM of recipient mice 20 days after transfer. (C) Ratios of c-MYC–expressing Tfap4^+/− to WT, Tfap4^+/− Erg^+/− to WT, and Erg^+/− to WT cells in the BM of recipient mice 20 days after transfer. (D) Percentages of surface IgM⁺ transduced Tfap4^+/+ B220⁺ cells compared with Tfap4^+/− B220⁺ cells in the same recipient mouse. (E) Percentages of CD45.2⁺ cells in the BM of mice in (A). (F) Percentages of surface IgM⁺ transduced Tfap4^+/− B220⁺ cells compared with Tfap4^+/− Erg^+/− or Erg^+/− B220⁺ cells (WT + Tfap4^+/−, n = 21; WT + Tfap4^+/− Erg^+/−, n = 23; WT + Erg^+/−, n = 9), Data are from 3 independent experiments combined. *P < .05, **P < .01, ****P < .0001; paired Student t test (C-D), **P < .01, ****P < .0001; Kruskal -Wallis test with Dunn’s multiple-comparison test (E,F). ns, no statistical significance.