Figure 5.
Gata2 acts as a tumor suppressor in mouse APL. (A) Leukemia-free survival of recipient Ly5.1 mice transplanted with nonleukemic lineage-depleted PML-RARA × Cas9 bone marrow in which Rosa26 intron 1 or Gata2 exon 2 was targeted with CRISPR guide RNAs prior to transplantation (n = 20 mice in Rosa26 group from 2 cohorts of 10 mice per cohort; n = 25 mice in Gata2-targeted PML-RARA × Cas9 bone marrow group from 3 cohorts of 5 to 10 mice per cohort; n = 6 mice in Gata2-targeted WT Cas9 bone marrow group from 1 cohort of mice). P < .0001, log-rank (Mantel-Cox) test. (B) Protein expression, detected using a Jess by ProteinSimple apparatus, of GATA2 and β-ACTIN from the bone marrow of Gata2-sufficient (Gata2+/+) or Gata2-targeted (Gata2−/−) Ctsg-PML-RARA APL mice. Blots for GATA2 and β-ACTIN were performed on the same samples using 2 antibodies in 2 spectral channels: chemiluminescence (GATA2) and near infrared (β-ACTIN). Data are representative of 3 or 4 independent biological replicates per genotype. (C) Quantification of the relative GATA2/β-ACTIN protein expression from the bone marrow of Gata2+/+ or Gata2−/−Ctsg-PML-RARA APL mice. Data are from 3 or 4 independent biological replicates per genotype. (D) CD34 and Gr-1 expression by flow cytometry on bone marrow from 8- to 12-week-old WT mice, Gata2+/+Ctsg-PML-RARA APL spleen cells, and Gata2−/−Ctsg-PML-RARA APL spleen cells. Data are representative of 3 or 4 independent biological replicates. (E) Wright-Giemsa staining of blood, bone marrow, and spleen from Gata2+/+ (Rosa26-targeted) or Gata2−/− (Gata2-targeted) Ctsg-PML-RARA APL mice. Images are representative of 5 to 8 independent biological replicates for each genotype. Images were captured using an Olympus BX-53 microscope and an Olympus DP-72 camera (original magnification ×100). (F) Leukemia-free survival of recipient Ly5.1 mice transplanted with fully transformed Gata2−/−Ctsg-PML-RARA APL spleen cells and then treated with surgically implanted ATRA pellets (black lines) or control pellets (Mock; red lines). N = 12 control-treated mice; N = 15 ATRA-treated mice. Gata2−/− APLs were derived from 3 biologically independent mice. P = .0003, log-rank (Mantel-Cox) test. (G) Leukemia-free survival of recipient Ly5.1 mice transplanted with Gata2+/+Ctsg-PML-RARA APL spleen cells and then treated with surgically implanted ATRA pellets (black lines) or control pellets (Mock; blue lines). N = 3 control-treated mice, N = 3 ATRA-treated mice. Gata2+/+ APLs were derived from 1 mouse. P = .0246, log-rank (Mantel-Cox) test.

Gata2 acts as a tumor suppressor in mouse APL. (A) Leukemia-free survival of recipient Ly5.1 mice transplanted with nonleukemic lineage-depleted PML-RARA × Cas9 bone marrow in which Rosa26 intron 1 or Gata2 exon 2 was targeted with CRISPR guide RNAs prior to transplantation (n = 20 mice in Rosa26 group from 2 cohorts of 10 mice per cohort; n = 25 mice in Gata2-targeted PML-RARA × Cas9 bone marrow group from 3 cohorts of 5 to 10 mice per cohort; n = 6 mice in Gata2-targeted WT Cas9 bone marrow group from 1 cohort of mice). P < .0001, log-rank (Mantel-Cox) test. (B) Protein expression, detected using a Jess by ProteinSimple apparatus, of GATA2 and β-ACTIN from the bone marrow of Gata2-sufficient (Gata2+/+) or Gata2-targeted (Gata2−/−) Ctsg-PML-RARA APL mice. Blots for GATA2 and β-ACTIN were performed on the same samples using 2 antibodies in 2 spectral channels: chemiluminescence (GATA2) and near infrared (β-ACTIN). Data are representative of 3 or 4 independent biological replicates per genotype. (C) Quantification of the relative GATA2/β-ACTIN protein expression from the bone marrow of Gata2+/+ or Gata2−/−Ctsg-PML-RARA APL mice. Data are from 3 or 4 independent biological replicates per genotype. (D) CD34 and Gr-1 expression by flow cytometry on bone marrow from 8- to 12-week-old WT mice, Gata2+/+Ctsg-PML-RARA APL spleen cells, and Gata2−/−Ctsg-PML-RARA APL spleen cells. Data are representative of 3 or 4 independent biological replicates. (E) Wright-Giemsa staining of blood, bone marrow, and spleen from Gata2+/+ (Rosa26-targeted) or Gata2−/− (Gata2-targeted) Ctsg-PML-RARA APL mice. Images are representative of 5 to 8 independent biological replicates for each genotype. Images were captured using an Olympus BX-53 microscope and an Olympus DP-72 camera (original magnification ×100). (F) Leukemia-free survival of recipient Ly5.1 mice transplanted with fully transformed Gata2−/−Ctsg-PML-RARA APL spleen cells and then treated with surgically implanted ATRA pellets (black lines) or control pellets (Mock; red lines). N = 12 control-treated mice; N = 15 ATRA-treated mice. Gata2−/− APLs were derived from 3 biologically independent mice. P = .0003, log-rank (Mantel-Cox) test. (G) Leukemia-free survival of recipient Ly5.1 mice transplanted with Gata2+/+Ctsg-PML-RARA APL spleen cells and then treated with surgically implanted ATRA pellets (black lines) or control pellets (Mock; blue lines). N = 3 control-treated mice, N = 3 ATRA-treated mice. Gata2+/+ APLs were derived from 1 mouse. P = .0246, log-rank (Mantel-Cox) test.

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