Restoration of Gata2 expression inhibits PML-RARA-induced aberrant self-renewal and leukemogenesis. (A) Retroviral “addback” of Gata2 in preleukemic PML-RARA–expressing cells. The initial protein expression detected using a Jess apparatus by ProteinSimple of GATA2 and β-ACTIN from preleukemic lineage-depleted PML-RARA × Cas9 bone marrow cells that were electroporated with CRISPR guide RNAs targeting Rosa26 intron 1 or Gata2 exon 2, serially replated in MethoCult M3534 for 8 weeks, and then transduced with empty vector, Gata2^WT, or Gata2^R362G MSCV-IRES-mCherry retroviruses. Samples are from cells that were grown in suspension culture with SCF, FLT3L, interleukin-3 (IL-3), and TPO (to minimize clonal selection pressures) for 3 days posttransduction, and then flow purified on mCherry⁺ cells. The Rosa26-targeted samples were run on a separate blot from the Gata2-targeted samples. Blots for GATA2 and β-ACTIN were performed using the same samples with 2 antibodies in 2 spectral channels: chemiluminescence (GATA2) and near infrared (β-ACTIN). (B) Fold change in the percentage of mCherry⁺ cells by flow cytometry in Gata2 exon 2–targeted PML-RARA × Cas9 bone marrow cells from panel A that were serially replated in MethoCult for 8 weeks, then retrovirally transduced with Gata2^WT, Gata2^R362G, or empty MSCV-IRES-mCherry vectors, and replated in MethoCult for an additional 2 to 3 weeks. Fold change is relative to the mCherry expression at day 3 posttransduction (n = 3 at days 7 and 14; n = 2 at day 21; independent biological replicates). *P < .05, **P < .01, ***P < .001,****P < .0001; 2-way ANOVA . (C) Retroviral “addback” of Gata2 in Gata2^−/− APLs. Protein expression was detected using a Jess apparatus by ProteinSimple of GATA2 and β-ACTIN at day 3 following the transduction of Gata2^−/− APL cells with empty vector, Gata2^WT MSCV-IRES mCherry retroviruses, or untransduced Gata2^+/+ APL cells. Cells were grown in suspension culture ex vivo with SCF, FLT3L, IL-3, and TPO (to minimize clonal selection pressures) for 3 days posttransduction. At day 3 posttransduction, APL cells were transplanted into sublethally irradiated Ly5.1 mice. Antibodies against GATA2 and β-ACTIN were used in the same samples in 2 spectral channels: chemiluminescence (GATA2) and near infrared (β-ACTIN). (D) Quantification of the relative GATA2/β-ACTIN protein expression in untransduced Gata2^+/+ APLs (green bar), empty vector–transduced Gata2^−/− APLs (blue bar), or Gata2^WT MSCV–transduced Gata2^−/− APLs (red bar) from the blots in panel C. **P < .01, ***P < .001; 2-way ANOVA. (E) mCherry expression by flow cytometry in Gata2^−/− APL samples transduced with empty vector (upper panels) or Gata2 MSCV-IRES-mCherry (lower panels) retroviruses. Samples are from APLs that were grown ex vivo for 3 days posttransduction with cytokines (left panels) or those that were subsequently transplanted into Ly5.1 mice and harvested on day 28 (right panels). Day-28 samples are gated on CD45.2⁺ peripheral blood APL cells. (F) Leukemia-free survival of recipient Ly5.1 mice that were transplanted with unsorted mCherry⁻ and mCherry⁺Gata2^−/−Ctsg-PML-RARA APL cells following transduction with empty vector or Gata2 MSCV-IRES-mCherry retroviruses. P = .6158; log-rank (Mantel-Cox) test. (G) Quantification of mCherry expression at day 3 and 28 in empty vector–transduced (left panel) and Gata2^WT MSCV–transduced (right panel) APLs from panel D. In panels E-G, each of 3 independently derived Gata2^−/− APLs was transduced with empty vector or Gata2 MSCV retroviruses and then transplanted into 5-6 Ly5.1 recipient mice (18 recipient mice received empty vector–transduced Gata2^−/− APLs; 17 recipient mice received Gata2 MSCV-mCherry–transduced Gata2^−/− APLs). ****P < .0001; unpaired, 2-tailed Student t test. NS, not significant.