Fig. 4

CAMKs play critical roles in human AML. a–d LILRB2 and CAMK expression negatively correlates with the overall survival of AML patients. The mRNA expression data of LILRB2 and CAMKs were obtained from the TCGA AML database (https://tcga-data.nci.nih.gov/docs/publications/tcga/; accessed November 5, 2012) and normalized by GADPH expression. Patients were separated into two groups based on whether they have higher (n = 83) or lower (n = 82) than the average expression levels of the indicated genes (n = 186). e An in silico analysis of human Camk1 mRNA expression in 43 human AML samples as described previously [24]. f Treatment with shRNAs targeting Camk1 or Camk4 inhibited the growth of MV4-11 cells. GFP⁺ cells were sorted by flow cytometry 1 day post-infection, and 20,000 cells were plated. Cell numbers were determined at three time points (days 2, 4, and 6) from triplicate wells. The experiment was repeated three times with similar results. g Inhibition of Camk1 or Camk4 expression inhibited the growth of KASUMI-1 cells. Cell numbers were determined at three time points (days 2, 4, and 6) from triplicate wells. The experiment was repeated three times with similar results. Knockdown of Camk1 and Camk4 in MV4-11 cells and KASUMI-1 cells as determined by Western blotting (h, i). k Rescue of Camk1-knockdown phenotype. Seven mutations were introduced into puromycin-seleted Camk1 expression lentivirus vector. Expression from this mRNA did not change CAMK1 amino acid sequence and was not silenced by shRNA Camk1. Mutant Camk1 (7 m) infected MV4-11 cells were resistant to the shRNA-Camk1-induced growth inhibition. The experiment was repeated three times with similar results. GFP⁺ cells were sorted by flow cytometry 1 day post-infection, and 20,000 cells were plated. Cell numbers were determined on days 2, 4, and 6 in triplicate wells. The experiment was repeated three times with similar results. l Western blot showed mutant Camk1 could not be silenced by shRNA Camk1. m Cell growth of human AML cells (U937, Kasumi1 and MV4-11) were inhibited upon CAMK (STO609) or CAMKK inhibitor (KN93) treatment. Numbers of cells were calculated 6 days post treatment; *p < 0.05