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. Author manuscript; available in PMC: 2019 Aug 4.
Published in final edited form as: Nat Cell Biol. 2019 Apr 22;21(5):640–650. doi: 10.1038/s41556-019-0314-5

Figure 3. IRAK4-L is required for leukemic cell function.

Figure 3.

(A) Overview of experimental design. (B) Colony formation of THP1 cells expressing shIRAK4-L, shIRAK4-L/S, or a control shRNA (shControl) (three independent experiments). Two-sided t-tests were used for statistical analyses. (C-D) THP1 cells expressing shIRAK4-L (n = 9 animals), shIRAK4-L/S (n = 5 animals), or a control shRNA (shControl; n = 6 animals) were engrafted into sublethally-irradiated NSG mice. After 6 weeks, leukemic burden (GFP+ cells) in the BM (C) and PB (D) was determined. Two-sided t-tests were used for statistical analyses. (E) Cord-blood CD34+ cells were transduced with shIRAK4-L, shIRAK4-L/S, or a control shRNA (shControl) and examined for IRAK4 knockdown (left; two independent experiments) and progenitor colony formation in methylcellulose (right; six independent experiments). Two-sided t-tests were used for statistical analyses. (F) THP1 cells transduced with empty retroviral vector (MSCV-IRES-GFP as a control) or THP1 IRAK4-KO cells transduced with retroviral vectors encoding IRAK4-L (left panel) or IRAK4-S (right panel) and then immunoblotted with the N-terminal IRAK4 antibody (left panel) and C-terminal IRAK4 antibody (right panel). (G-H) Representative images of parental and IRAK4-KO THP1 cells examined for leukemic progenitor function in methylcellulose (three biological replicates). Scale bar, 700 microns. Two-sided t-tests were used for statistical analyses. (I) Colony formation of MDS/AML cell lines and control CD34+ cells (2 independent donors) treated with DMSO or CA-4948 for 7–10 days (three independent experiments). Two-sided t-tests were used for statistical analyses. (J) Graph representing the IC50 relative to the ratio of IRAK4-L to IRAK4-S expression in the indicated cell lines and CD34+ cells summarized from Figure 1I and 3I. (K) THP1 cells were xenografted in NSG mice (n = 10) and treated with IRAK4 inhibitor (CA-4948) or vehicle at 12.5 mg/kg/5d/week for 5 weeks. (L) Mice xenografted with THP1 cells were assessed for leukemic engraftment in BM, and spleen and liver weight. Two-sided t-tests were used for statistical analyses. (M) Representative spleens are shown isolated from mice xenografted with THP1 cells at time of death after treatment with vehicle or CA-4948. All data represent the mean ± s.e.m.