Fig 2: CREB activity plays important role in PirB/LILRB2-CAMKs signaling in AML-SCs. a Phosphorylation of CREB was decreased in the PirBTM MLL-AF9 AML BM cells compared to control cells. b–d Retrovirally expressed Creb, but not Creb mutant (S129A), rescued PirBTM phenotype upon secondary transplantation. Retrovirally expressed Creb had similar levels as endogenous proteins in WT controls. b Survival curves of mice transplanted with 3000 of these ectopically Creb-expressing, Creb S129A-expressing, or control cells (n = 10 mice). c Percentages of retrovirus-infected (GFP+) AML cells in PB of secondary recipient mice after 28 days of transplantation. (n = 5 mice). d CFU numbers of retrovirus-infected (GFP+) AML cells in colony-forming assays. The experiment was repeated three times with similar results; e CAMKI and LILRB2 bound in transfected 293T cells. The indicated flag-tagged LILRB2 or myc-tagged CAMKI or CAMKIV proteins were overexpressed in 293 cells. Flag antibody was used to precipitate LILRB2 proteins, and the flag or myc antibodies were used in Western blots. f Endogenous CAMKI and LILRB2 interact with each other as determined by bidirectional pull-down assays. MV4-11 cells (1 × 107 cells) were lysed with transmembrane protein extraction reagent and indicated antibodies were used for immunoprecipitation and Western blot; *p < 0.05. g Schematic summary of the signaling pathway mediated by the PirB/LILRB2-CAMKs-CREB in AML cells

Fig 3: Loss of CAMKIV decreases self-renewal capacity of AML-SCs. a, b Representative flow cytometry plots show that Camk4-null MLL-AF9 AML mice had decreased numbers of YFP+Mac-1+Kit+ progenitors in both primary and secondary transplants. c, d Primary and secondary transplants with Camk4-null MLL-AF9 BM cells show significantly decreased colony-forming ability (n = 3). e Camk4-null MLL-AF9 BM cells show increased apoptosis. AnnexinV+/7AAD- indicates early apoptosis, and AnnexinV+/7AAD+ indicates late apoptosis. *p < 0.05. f Limiting dilution assays comparing the frequencies of AML stem cells in WT and Camk4-null MLL-AF9 AML. The indicated YFP+ WT and Camk4-null MLL-AF9 BM cells that were collected from primary recipients were co-transplanted with 2 × 105 bone marrow competitor cells into lethally irradiated recipients. The CRUs were calculated by L-Calc software

Fig 4: Effects of G-Re on LPS-induced activation of CAMKs and mitogen-activated protein kinases. BV2 cells were incubated with KN93 (5 µM), KN92 (5 µM), PD98059 (5 µM) or SP600125 (5 µM), followed by LPS (1 µg/ml) treatment for 24 h. (A) Nitric oxide content was measured, and (B) protein expression levels of iNOS and COX-2 were detected by western blotting. (C) Relative intensity of each band (normalized to ß-actin) was indicated as a ratio to control. BV2 cells were treated with various concentrations of G-Re for 1 h, followed by LPS treatment (1 µg/ml) for 30 min. (D) Expression levels of unphosphorylated kinases or p-kinases were analyzed by western blotting. (E-H) Relative intensity of p-kinases (normalized to respective unphosphorylated kinases) was indicated as a ratio to control. *P<0.05 vs. the group treated with LPS alone. CAMK, Ca2+/calmodulin-dependent protein kinase; COX-2, cyclooxygenase 2; ERK, extracellular signal-regulated kinase; G-Re, ginsenoside Re; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinases; LPS, lipopolysaccharide; p, phosphorylated.

Fig 5: Knockdown of CAMK1 or CAMK4 blocks xenograft of human leukemia cells. a Survival curve of NSG mice transplanted with MV4-11 cells (1 × 106 cells) infected with virus designed to express GFP and either scrambled shRNA, Camk1 shRNA, or Camk4 shRNA. GFP+ cells were collected and transplanted into NSG mice 1 day post-infection (n = 10 mice). b Percentages of GFP+ cells in BM, spleen, liver, and PB at 28 days after transplantation. c Representative flow cytometry plots indicating decreased spleen engraftment of MV4-11 cells treated with shRNA targeting Camk1 or Camk4. Staining with anti-human CD45 antibodies confirmed engraftment was from transplanted human leukemia cells. The percentages of hCD45+ population are indicated. d Comparison of the sizes of spleens of the mice transplanted with control MV4-11 leukemia cells or cells expressing shRNA targeting Camk1 or Camk4 (n = 5 mice); *p < 0.05