Fig. 6.

aPKCι is required for the survival and maintenance of human CML CD34⁺ cells. a Schematic diagram of shRNA mediated knockdown of aPKCι in CML patient-derived CD34⁺ cells followed by in vitro culture as well as in vivo transplantation into NSG-S mice. b Q-RT-PCR showing shRNA mediated knockdown of aPKCι in CML CD34⁺ cells. c Colony forming unit (CFU) cell assay showing reduced clonogenic efficiency of aPKCι deficient CML CD34⁺ cells. d Liquid culture expansion of control and aPKCι deficient CML CD34⁺ cells. e FACS quantification of CD34⁻/CD11b⁺ cells derived from control and aPKCι knocked down CML CD34⁺ cells grown in liquid culture medium. f FACS quantification of annexin V-binding of control/aPKCι knocked down CML CD34⁺ cells growing in liquid culture expansion medium. g hCD45⁺/EGFP⁺ chimera in NSG-S mice transplanted with CML CD34⁺ cells transduced with control/aPKCι shRNA lentiviruses. h, i Quantification of BrDU uptake (h) and annexin V-binding (i) of hCD45⁺/EGFP⁺ chimera in the bone marrow (BM) of NSG-S mice transplanted with control/aPKCι shRNA transduced CML CD34⁺ cells. j hCD45⁺/hCD11b⁺ on EGFP⁺ chimera in NSG-S mice transplanted with CML CD34⁺ cells transduced with control/aPKCι shRNA lentiviruses. k Schematic diagram of pharmacological administration of imatinib and a pan-PKC inhibitor (Ro-31-8200) to NSG-S mice transplanted with CML CD34⁺ cells and T315I p210 BCR-ABL transduced CB CD34⁺ cells. l FACS quantification of human CD45⁺ cell engraftment in the BM of NSG-S mice transplanted with CML CD34⁺ cells and treated with either control vehicle, imatinib, Ro-31-8200 or a combination of imatinib and Ro-31-8200. Imatinib and Ro-31-8200 administration led to a similar decrease of human CML burden in the BM of NSG-S mice. m FACS quantification of hCD45⁺/EGFP⁺ chimera in the BM of NSG-S mice transplanted with T315I transduced CB CD34⁺ cells and treated with control vehicle, imatinib or R0-31-8200. Mice treated with the PKC inhibitor show reduced levels of human CD45⁺/EGFP⁺ chimera in the BM, whereas imatinib treatment has no effect. Cont: Control. Data are presented as mean ± SD of a minimum of two independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001, t-test