Pifithrin-α hydrobromide (Pifithrin-α hydrobromide) is a p53 inhibitor, inhibiting p53-dependent transactivation of p53-responsive genes.

The ligand binding competition assays are performed. Cytosolic cell extracts from Hepa-1 cells are generated by the resuspension of the cell pellets in HEDG buffer [25 mM Hepes, 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol, pH 7.5] containing 0.4 mM leupeptin, 4 mg/mL aprotinin, and 0.3 mM phenylmethylsulfonyl fluoride, homogenization, and centrifugation at 100,000 g for 45 min. Aliquots of the supernatant (120 μg) are incubated at room temperature for 2 h with the indicated concentrations of Pifithrin-α in the presence of 3 nM [3H]TCDD in HEDG buffer. After incubation on ice with hydroxyapatite for 30 min, HEDG buffer with 0.5% Tween 80 is added. The samples are centrifuged, washed twice, resuspended in 0.2 mL of scintillation fluid, and subjected to scintillation counting. Nonspecific binding is determined using a 150-fold molar excess of TCDF and subtracted from the total binding to obtain the specific binding. The specific binding is reported relative to [3H]TCDD alone[2].

At the end of cell treatments, the number of attached cells is estimated by staining with 0.25% crystal violet in 50% methanol, followed by elution of the dye with 1% SDS. Optical density (530 nm) reflecting the number of stained cells is determined with a Bio-Tek EL311 microplate reader. Cell viability in suspension of short term culture of primary thymocytes is determined by their staining with 0.1% of methyl blue and microscopic counting of blue (dead) cells.(Only for Reference)

DMSO: 50 mg/mL (136.13 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (5.45 mM), Sonication is recommended.