Statistical significance was calculated by one-way ANOVA with Dunnetts correction for multiple comparison

Statistical significance was calculated by one-way ANOVA with Dunnetts correction for multiple comparison. as a potential novel niche-based strategy to improve the efficacy of AML therapy. ability of malignant cells to bind biological lectins like E-selectin. Thus, herein we investigate the functional roles of CD44 and CD162 as E-selectin ligands on AML cells in human and preclinical mouse models. We show that INH6 CD162, but not CD44, is required for E-selectin-mediated chemo-resistance and INH6 report a novel role for CD162 in AML progression and response to treatment = 17 recipients of = 5 mice/group. Statistical analysis performed by unpaired two-tailed = 5C9 mice/group). Statistical significance was calculated by unpaired two-tailed Chemo-Sensitivity Assay Tissue culture wells (96-well plates) were coated with recombinant human PECAM-1/CD31, P-selectin, E-selectin and CD14 (non-adhesion control) human IgG1 Fc fusion proteins (purchased from R&D Systems) at a concentration of 5 g/mL in 30 L of TrisCHCl 20 mM pH 8.6, overnight at 4C. Non-coated control wells were also included. The next Rabbit polyclonal to CD3 zeta day, unbound proteins were removed and the wells were washed with PBS before being blocked for one hour in X-VIVO media at 37C. Ten thousand KG1a cells were then added into each well, in a volume of 100 L of X-VIVO. The plate was incubated for 7 h at 37C before Ara-C or saline control was added in 20 L at final concentration of 10 g/mL, 100 g/mL, or 1 mg/mL. After 48 h of incubation at 37C, cell viability was assessed using the CellTox Green Cytotoxicity assay and CellTiter-Glo INH6 2.0 viability assay (both from Promega) on a PHERAstar microplate reader (BMG Labtech) according to the manufacturers instructions. Two negative (non-adhesion) controls were included for chemo-sensitivity assay, (1) parallel blocked non-coated wells, and (2) parallel control wells coated with recombinant human CD14 IgG1 Fc fusion protein (as non-adhesion control). Generation of Receptor Deleted Human AML Cell Line by CRISPR Gene Editing CRISPR-Cas9 gene editing was used to delete and genes from KG1a cells. Design of the single guide (sg) RNAs and optimization were based on a protocol developed by Charles C. Bell in Andrew Perkins group (Bell et al., 2014) (ACBD, Monash University, Australia) as well as published protocol by Ran et al. (2013). Deskgens CRISPR guide INH6 RNA design tool1 was used to generate single guide (sg) RNAs sequences with minimal exonic off-target sites. Two sgRNAs were designed for both and (each targeting a different exon), with target sequences as follows: 5-TCGCTACAGCATCTCTCGGA and 5-TGGGTTCATAGAAGGGCACG for The sgRNAs were cloned into the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene cat# 48138) after Bpil digestion. Purified plasmids were transfected in KG1a cells using the 4D-NucleofectorTM (Lonza) according to the manufacturers instructions with the DI-100 setting. At day 6 post nucleotransfection, cell surface staining (using anti-human conjugated antibodies CD44-APC and CD162-PE, see Supplementary Table 1) revealed the INH6 appearance of a small population of cells that were negative for either CD44 or CD162. The next day, single cells from these populations were sorted using BD FACSAriaTM Fusion (BD Biosciences) into 96-well plates and cultured in MEM supplemented with 10% FCS and 1X penicillin-streptomycin-glutamine (all from Gibco). Around 4 weeks later, several KG1a clones emerged from these single cells and were again tested by flow cytometry for cell surface expression of CD44 and CD162. Clones that displayed >99% cells negative for CD44 or CD162 (as shown in Figure 1D) were selected and expanded for use in studies. Control WT KG1a cells were subjected to the same process as transfected KG1a cells except for the omission of sgRNA. Open in a separate window FIGURE 1 CD44 and CD162 co-localize with E-selectin on human AML cell surface.