Figure 2Functional role of GPR56 in vitro. Left: CB CD34+ cells were transduced with shGPR56weak and shGPR56strong versus shLuc as negative control. The loss of the GFP+ fraction on day 8 and day 11 compared with day 3 post transduction (delta GFP+) correlates with the level of GPR56 KD. Three replicate wells were monitored per condition, unpaired t-test, bars and error bars represent mean and standard deviation, *** p<0.0005, ** p<0.005, * p<0.05. Right: Colony forming cell assay. Pooled CB CD34+ cells were transduced with shRNAs against GPR56 or shLuc control, and GFP+ cells were directly sorted into methylcellulose 72 hours post transduction. 250 GFP+ cells were plated per well and colonies were counted 10 days post plating. CFC: colony forming cell, CFU: colony forming unit, G: granulocyte, M: macrophage, GM: granulocyte/macrophage, E: erythrocyte, GEMM: granulocyte, erythrocyte, megakaryocyte, macrophage. Shown is one of two independent assays. 6 wells were plated per condition, unpaired t-test, bars and error bars represent mean and standard deviation. * p<0.05, ** p<0.005. Functional role of GPR56 in vivo. CB CD34+ cells were transduced with the two shRNAs against GPR56 versus shLuc as negative control. Gene transfer was approximately 50% at experiment start. Equal numbers of cells were injected in 10 recipient NSG mice per condition and engraftment of human CD45+ (huCD45) cells of bone marrow was assessed at 4 and 8 weeks by bone marrow aspiration and at 20 weeks by total bone marrow harvest. Strong GPR56 KD (shGPR56strong) significantly reduced overall human CD45+ engraftment, while incomplete KD (shGPR56weak) showed a trend towards lower engraftment at 4 weeks, but no difference at later time points indicating that low GPR56 surface levels might be sufficient for LT-HSC engraftment. Unpaired t-test of log10-transformed values, symbols represent individual mice, horizontal bars represent geometric means, BM: bone marrow. Violin plots showing geometric mean (horizontal line) and individual values (circles) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34+ cells following overnight-infection with shGPR56strong (turqouise) and shGPR56weak (gray) versus shLuc control (violet). Shown are percentages of indicated populations, which are also co-positive for human CD45 and GFP, relative to all harvested bone marrow cells. HSPC: CD34+SSClowCD33-CD19-, B precursor: CD34+CD19+CD33-, myeloid progenitor (myelo. prog.): CD34+CD33+CD19-, mature B cells: CD19+CD34-, mature myeloid cells: CD33+CD19-CD34-. Multiple unpaired t-tests of log-transformed values, p-values were Benjamini and Hochberg corrected (padj), * padj<0.05, ** padj<0.005, *** padj<0.0005. Log2-fold changes (log2FC) of the CD19+ (B lymphoid) versus CD33+ (myeloid) cell ratios within the human CD45+GFP+ fractions at final analysis 20 weeks after transplantation. Both shRNAs change the lymphoid/myeloid ratio in favor of myeloid cells. Unpaired t-test of log2FC, symbols represent individual mice, horizontal bars represent average log2FC, * p<0.05. GPR56 KD results in highly reduced engraftment of AML-491 in mice. Left: Cells from two independent infections with shGPR56strong were injected in 4 NRGS mice. Shown are fractions of GFP+ cells before and 10 weeks after transplantation (Tx). Unpaired t-tests of log(%GFP+) before versus after transplantation. Right: weak GPR56 KD results in highly reduced engraftment of AML-491 in NSGW41 mice. Cells were injected in 4 recipient NSGW41 mice. Shown are fractions of positively transduced Ametrine (AM)+ cells before and 32 weeks after Tx. Unpaired t-tests of log(%AM+) before versus after transplantation. No difference in overall leukemic engraftment (including non-transduced cells) was observed excluding technical issues with injections Adhesion of control and GPR56 KD K562 cells on fibronectin-functionalized substrates. Left: Overlay of phase contrast and microinterferometry images of control (above) and GPR56 KD cells (below). The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet, respectively. Right: Comparison of adhesion areas extracted by microinterferometry, reduction of adhesion area by factor 1.9 in GPR56 KD cells (p = 1.0 x 10-10, two-sided Mann-Whitney test, box plots showing medians, quartiles, and outliers according to the Tukey method). Technical replicates Ncontrol = 71, Technical replicates NKD = 104, scale bar 10 µm. Active deformation of control and GPR56 KD cells. Left: Cell periphery of control (top) and GPR56 KD (bottom) K562 cells tracked over 60 min. Right: Comparison of deformation power of control and GPR KD cells, showing a reduction of deformation power by factor 1.8 (p = 0.005, two-sided Mann-Whitney test, white triangles of the violin plot represent mean). Technical replicates Ncontrol = 40, Technical replicates NKD = 40, scale bar 10 µm.