Z
Z., Y. compound suramin efficiently inhibits IP5K and and and and and Table 1). Suramin, an anti-parasitic drug approved by the World Health Organization (35), is by far the most potent IP5K inhibitor, with an apparent IC50 of 2.6 m based on the ADP-Glo assay (Fig. 1value is well-aligned with suramin’s IC50 against IP5K (2.6 m; Fig. 1and and and Fig. S2value (1.5 m) comparable with the reported and and and < 0.05; **, < 0.01 (Student's test). < 0.01 (Student's test). and then stained with FITC-conjugated annexin V and PI before being analyzed by flow cytometry. + and Fig. 1and and < 0.01; ***, < 0.001 (Student's test). and and < 0.05; **, < 0.01 (Student's test). The above results led us to hypothesize that NF449 would bind IP5K similarly to suramin, which we probed by docking analysis (Fig. 4and Fig. S4= Bottom + (Top ? Bottom)/[1 + (for 5 min at 4 C. At this time, we prepared the TiO2 beads (Titansphere TiO 5 m; GL Sciences), which were weighed and prepared by washing once in water then once in 1 m PA containing 5 mm EDTA, (4C5 mg for one sample). The supernatants were removed into new Eppendorf tubes, and TiO2 beads were added. The samples were rotated for 15 min at 4 C. The beads were pelleted by centrifuging at 3500 for 1 min and then washed twice in PA with the supernatants discarded. Bound inositol phosphates were then eluted with 200 l of 5% ammonium hydroxide. The eluents were then vacuum-evaporated to 50 l and subjected to 35.5% PAGE. Phosphate-rich metabolites were imaged with toluidine blue staining, using commercial IP6 as a control. Western blotting and coimmunoprecipitation HEK293 cells stably expressing myc-CSN2 were as described before (12). For CRLCCSN interaction analysis, the cells were treated with 20 m suramin or 10 m NF449 for 8 h, with normal HEK 293 cells as control. Myc immunoprecipitation experiments were performed as previously described (57). The samples were loaded to SDS-PAGE gel for Western blotting of the indicated proteins. Where applied, IP5K was knocked down in myc-CSN2 stable HEK293 cells using reagents as previously described (11). Cell viability assay HCT116 cells were seeded to 24-well plates with 8 104/well and were treated with various drug or drug combinations for 48 Cholic acid h to test the viability of cells by counting. The results Cholic acid were normalized to untreated control wells. Cell cycle analysis HCT116 cells were Cholic acid plated in 6-well plate and incubated with 0.5 m MLN4924, 20 m suramin, or 0.5 m MLN4924 plus 20 m suramin for 24 h. After trypsinization (0.25% trypsin without EDTA), the cells were washed with cold PBS and fixed with 70% ethanol overnight at 4 C. Rabbit Polyclonal to ARMCX2 Fixed cells were centrifuged at 1000 rpm for 5 min to remove ethanol and then resuspended by 50 g/l propidium iodide (BD, 550825) and 50 g/l RNase in PBS for 30 min on ice protected from light. Cell cycle distributions were determined by flow cytometry (BD FACSCanto) and analyzed using FlowJo 10. Apoptosis (annexin VCPI) analysis HCT116 cells were plated in 6-well plate overnight and incubated with 0.5 m MLN4924, 20 m suramin, or 0.5 m MLN4924 with 20 m suramin for 24 h, respectively. After trypsinization, the cells were washed twice with cold PBS and resuspended in 1 binding buffer at a concentration of 1 1 106 cells/ml. 1 105 cells were stained with FITCCannexin V and propidium iodide Cholic acid (BD, 556547) for 15 min at room temperature protected from light. After the addition of 400 l of binding buffer, the cells were sorted by flow cytometry (BD FACSCanto), and the.