![GATA2 silencing phenocopies HMGA1 silencing in preclinical models of JAK2V617F MPN AML. (A) Relative GATA2 expression (mean ± standard deviation [SD]) from 2 experiments performed in triplicate (quantitative polymerase chain reaction) in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs GATA2-sh1 or GATA2-sh2). GAPDH was used as a loading control. (B) Western blots of GATA2, β-actin, and GAPDH (loading control) protein levels from DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2). Western blots were performed 3 times with similar results; a representative blot is shown. Size markers (kDa) are indicated. (C) Proliferation (mean ± SD) by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega)] assay in DAMI, SET-2 and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2), at the indicated time points. (D) Colony-forming units (CFU; mean ± SD) in DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2), in 2 experiments performed in triplicate. Representative images of colony formation after silencing GATA2 by delivery of a lentiviral vector expressing shRNA targeting GATA2 (control vs GATA2-sh1 or GATA2-sh2) are shown. Scale bars, 200 μm. (E) Edu (5-ethynyl-2′-deoxyuridine) cell cycle (mean ± SD) by flow cytometry assessed in DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2), in 2 experiments performed in triplicate. (F) Annexin V apoptosis by flow cytometry assessed in DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2) in 2 experiments. (G) Representative images of spleens and relative spleen size (mean ± SD); leukemia cell engraftment (mean ± SD) assessed by flow cytometry in spleen from NSG mice injected with DAMI cells with or without GATA2 silencing (control vs GATA2-sh1; n = 4 per group). (H) Leukemia cell engraftment assessed by flow cytometry (mean ± SD) in BM from NSG mice injected with DAMI cells, with or without GATA2 silencing (control vs GATA2-sh1; n = 4 per group). Representative flow cytometry plots from each group and hematoxylin and eosin–stained BM are shown. Scale bar, 200 μm. (I) Survival analysis by Kaplan-Meier estimate assessed in NSG mice injected with DAMI cells transduced with lentivirus (control: median survival, 24 days vs GATA2-sh1: median survival, 38.5 days; n = 6 per group). *P < .05, **P < .01, ***P < .001, ***P <.001, 2-tailed Student t test (A,C-H), log-rank (Mantel-Cox) test (I).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/139/18/10.1182_blood.2021013925/4/bloodbld2021013925f5.png?Expires=1736046951&Signature=Bbzr45cUA5n899IJXSDwP5AKepiefV1VUMRYgt9jOKrqATK-BvUxaHzdOG-K5IW1w2~dC5-8bt8vwhvch-7rz1y1j~Mldy5Y5irWJ~7t6yJkcdHeEsArig7YF~34isJ0WlXiVLsJ9n-3L6V8oF3LxXIeaVMzmg4M71qa~Usxt1jOwCRq2ixRSK-uufgZUbvWwr7k2Jei2B~RgczqRuNF1vZxYmhw8VitzqvFUbFcg2NVvAWICsgFYC9f~~PWeLBmIv1uz1JSy1dU~QQgZh9ev9e4oJPW3Ue-hTfL70mXo3qovsmZD7PDIu1ST3herta-i~KYexnHe2cFeT6uw6WYRw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
GATA2 silencing phenocopies HMGA1 silencing in preclinical models of JAK2V617F MPN AML. (A) Relative GATA2 expression (mean ± standard deviation [SD]) from 2 experiments performed in triplicate (quantitative polymerase chain reaction) in DAMI, SET-2, and UKE-1 cells, with or without HMGA1 silencing (control vs GATA2-sh1 or GATA2-sh2). GAPDH was used as a loading control. (B) Western blots of GATA2, β-actin, and GAPDH (loading control) protein levels from DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2). Western blots were performed 3 times with similar results; a representative blot is shown. Size markers (kDa) are indicated. (C) Proliferation (mean ± SD) by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Promega)] assay in DAMI, SET-2 and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2), at the indicated time points. (D) Colony-forming units (CFU; mean ± SD) in DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2), in 2 experiments performed in triplicate. Representative images of colony formation after silencing GATA2 by delivery of a lentiviral vector expressing shRNA targeting GATA2 (control vs GATA2-sh1 or GATA2-sh2) are shown. Scale bars, 200 μm. (E) Edu (5-ethynyl-2′-deoxyuridine) cell cycle (mean ± SD) by flow cytometry assessed in DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2), in 2 experiments performed in triplicate. (F) Annexin V apoptosis by flow cytometry assessed in DAMI, SET-2, and UKE-1 cells, with or without GATA2 silencing (control vs GATA2-sh1 or GATA2-sh2) in 2 experiments. (G) Representative images of spleens and relative spleen size (mean ± SD); leukemia cell engraftment (mean ± SD) assessed by flow cytometry in spleen from NSG mice injected with DAMI cells with or without GATA2 silencing (control vs GATA2-sh1; n = 4 per group). (H) Leukemia cell engraftment assessed by flow cytometry (mean ± SD) in BM from NSG mice injected with DAMI cells, with or without GATA2 silencing (control vs GATA2-sh1; n = 4 per group). Representative flow cytometry plots from each group and hematoxylin and eosin–stained BM are shown. Scale bar, 200 μm. (I) Survival analysis by Kaplan-Meier estimate assessed in NSG mice injected with DAMI cells transduced with lentivirus (control: median survival, 24 days vs GATA2-sh1: median survival, 38.5 days; n = 6 per group). *P < .05, **P < .01, ***P < .001, ***P <.001, 2-tailed Student t test (A,C-H), log-rank (Mantel-Cox) test (I).