CTX1

Kinase experiment:

Exponentially growing OCI cells cultures are treated with 3 μM CTX1, 8 μM Nutlin and or 15 μM RO-5963 for 4.5 hrs. Whole-cell extracts are generated using modified RIPA lysis buffer 25 mM Tris (pH 8.0), 100 mM NaCl, 0.5 mM EDTA, 0.50% NP-40 and complete protease mixture tablet. Protein extracts (~750 μg) are precleared and immune precipitation is performed using a kit according to the manufacturer's protocol. For the immunoprecipitation, mouse monoclonal anti-p53 and rabbit polyclonal anti-HDMX are used. Immune complexes are then collected, proteins are eluted and subjected to Western blotting with the indicated antibodies[1].

Animal experiment:

6 week old female NOD/SCID/IL2Rγ−/− mice are injected into the tail vein with 5×106 cells primary human AML cells (n=5 per group). Drug treatment is started 2 days after tumor cell injection. Nutlin-3 is given by oral gavage (200 mg/kg) and CTX1 is injected i.p. (30 mg/kg) five days a week for 3 weeks. Flow cytometry is performed on bone marrow cells isolated from the mouse femur using a human specific CD45PE antibody using a cytometer to confirm AML infiltration into the bone marrow[1].

References:

[1]. Karan G, et al. Identification of a Small Molecule That Overcomes HdmX-Mediated Suppression of p53. Mol Cancer Ther. 2016 Apr;15(4):574-582.