Figure 1.
Gadd45a is negatively regulated by Lgr4 and its deletion retains β-catenin activity even in the absence of Lgr4. (A) Heat map analysis of microarray data (n = 3, P ≤ .005, fold change ≥ 1.8) showing differentially expressed genes induced by Lgr4 knockdown in MLL-AF9–induced AML cells. (B) Quantitative polymerase chain reaction (qPCR) (n = 3) and Western blots confirming upregulation of Gadd45a induced by Lgr4 knockdown in MLL-AF9 leukemic cells carrying scrambled control (Scr) vs Lgr4 shRNA1 (sh1). Data are given as mean ± SD. ∗P < .05, unpaired t-test. (C) qPCR (n = 3) and Western blots showing downregulation of Gadd45a induced by Lgr4 overexpression in HOXA9/MEIS1 leukemic cells, compared with endogenous expression of Gadd45a in MLL-AF9 leukemic cells. Data are given as mean ± SD. ∗∗∗∗P < .0001, 1-way analysis of variance (ANOVA). (D) Analysis of TCGA dataset,21,22 revealing the correlation between LGR4 and GADD45A expression in all patients with AML (n = 244, r = −0.328, P = 1.6e-07), patients with AML with unfavorable outcome (n = 81, r = −0.482, P = 5.1e-06), and patients with AML with favorable outcome (n = 120, r = −0.155, P = .091). (E) Western blots confirming efficient Gadd45a knockout with a resultant increase in β-catenin and inactive phospho-Ser9-Gsk3β (p-Gsk3βSer9) in GFP+ pre-MLLc-Kit+ cells. qPCR showing upregulation of Wnt/self-renewal target genes induced by Gadd45a deletion (n = 3). Data are given as mean ± SD. ∗P < .05, ∗∗P < .005, unpaired t-test. (F) Colony formation of Gadd45a−/− vs Gadd45a+/+ pre-MLLc-Kit+ cells. The percentage of colonies (relative to Gadd45a−/−) at the third round of serial replating is shown. n = 4 independent experiments. Data are given as mean ± SD. ∗∗P < .005, unpaired t-test. (G) Western blots confirming efficient Lgr4 knockdown with a resultant change of endogenous β-catenin expression in response to Gadd45a knockout in LSCs (Lin–Sca-1–c-KithighCD16/32highCD34+), flow sorted from the bone marrow (BM) of AML mice following transplantation with pre-MLLc-Kit+ cells.

Gadd45a is negatively regulated by Lgr4 and its deletion retains β-catenin activity even in the absence of Lgr4. (A) Heat map analysis of microarray data (n = 3, P ≤ .005, fold change ≥ 1.8) showing differentially expressed genes induced by Lgr4 knockdown in MLL-AF9–induced AML cells. (B) Quantitative polymerase chain reaction (qPCR) (n = 3) and Western blots confirming upregulation of Gadd45a induced by Lgr4 knockdown in MLL-AF9 leukemic cells carrying scrambled control (Scr) vs Lgr4 shRNA1 (sh1). Data are given as mean ± SD. ∗P < .05, unpaired t-test. (C) qPCR (n = 3) and Western blots showing downregulation of Gadd45a induced by Lgr4 overexpression in HOXA9/MEIS1 leukemic cells, compared with endogenous expression of Gadd45a in MLL-AF9 leukemic cells. Data are given as mean ± SD. ∗∗∗∗P < .0001, 1-way analysis of variance (ANOVA). (D) Analysis of TCGA dataset,21,22 revealing the correlation between LGR4 and GADD45A expression in all patients with AML (n = 244, r = −0.328, P = 1.6e-07), patients with AML with unfavorable outcome (n = 81, r = −0.482, P = 5.1e-06), and patients with AML with favorable outcome (n = 120, r = −0.155, P = .091). (E) Western blots confirming efficient Gadd45a knockout with a resultant increase in β-catenin and inactive phospho-Ser9-Gsk3β (p-Gsk3βSer9) in GFP+ pre-MLLc-Kit+ cells. qPCR showing upregulation of Wnt/self-renewal target genes induced by Gadd45a deletion (n = 3). Data are given as mean ± SD. ∗P < .05, ∗∗P < .005, unpaired t-test. (F) Colony formation of Gadd45a−/− vs Gadd45a+/+ pre-MLLc-Kit+ cells. The percentage of colonies (relative to Gadd45a−/−) at the third round of serial replating is shown. n = 4 independent experiments. Data are given as mean ± SD. ∗∗P < .005, unpaired t-test. (G) Western blots confirming efficient Lgr4 knockdown with a resultant change of endogenous β-catenin expression in response to Gadd45a knockout in LSCs (Lin–Sca-1–c-KithighCD16/32highCD34+), flow sorted from the bone marrow (BM) of AML mice following transplantation with pre-MLLc-Kit+ cells.

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