Cell experiment:

The cell viability is determined by the Cell Counting Kit-8 assay. Briefly, A549 and H460 cells are incubated in 96-well plates at a density of 5 × 103 per 100 μL of culture medium overnight. After treated with indicated concentration of Pifithrin-μ for 24 and 48 h, 10 μL of tetrazolium substrate are added to each well of the plate. After incubation at 37℃ for 1 h, the absorbance is recorded at a wavelength of 450 nm using a microplate reader. Each experiment is determined in triplicate and repeated at least three times[2].

Animal experiment:

Mice[2]A549 cells (1 × 107) are suspended in Matrigel and inoculated subcutaneously into the mice. Twelve mice bearing evident tumors are arbitrarily assigned to PBS control group and Pifithrin-μ treatment groups (six mice per group). When tumors reach a size of ~5×5 mm2, mice are treated with either a single of intraperitoneal injection of Pifithrin-μ (20 mg/kg) or PBS every two days. After 3-week treatment, mice are euthanized with carbon dioxide. Tumor burdens are evaluated by measuring body weight, tumor weight, and tumor volume. Tumor volume is determined as 0.5 × length × width2. Tumor samples are collected and fixed in 10% neutral buffered formalin. Hematoxylin and eosin staining and immunohistochemistry for histological analysis of tumor samples are measured[2].

Pifithrin-μ is a potent inhibitor of  p53 binding and p53-mediated apoptosis with Kd value of 0.82 mM in vitro[1].
The p53 is encoded in humans by the TP53 gene. The molecular mass of p53 is 53 KD. The p53 has the function of regulating the cell cycle, thus, it functions as preventing cancer, a tumor suppressor. The p53 plays an important role in apoptosis, inhibition of angiogenesis and genomic stability by activating DNA repair proteins, arresting cell growth though holding the cell cycle and initiating apoptosis. p53 becomes activated in response to DNA damage, osmotic shock, oxidative stress or other myriad stressors. Activated p53 activates the expression of several genes by binding DNA including p21. p21 binds to the G1-S/CDK complexes which is an important molecules for the G1/S transition, then causes cell cycle arrest. The increasing amount of p53 may be a solution for prevention of tumors spreading or treatment of them[1].
Pifithrin-μis a cell-permeable inhibitor of p53-binding and p53-mediated apoptosis. Pifithrin-μdirectly inhibits that p53 binds to mitochondria. Pifithrin-μ also inhibits p53 binds to Bcl-2 and Bcl-xL proteins. PFT-μbinds both Bcl-xL and p53 with Kd = 0.80 mM and 0.82 mM respectively.[1] Pifithrin-μ reduces apoptosis which triggered by nutlin-3 in ML-1 cells at 25μM [2]. Pifithrin-μ also selectively inhibits heat shock protein 70 (HSP 70) activity.
Pre-treatment with Pifithrin-μcan rescue primary thymocytes from γ-irradiation  or DNA damaging agents in mice.[2]    
References:
[1].    Hagn F, Klein C, Demmer O, Marchenko N, Vaseva A, Moll UM, Kessler H: BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain. J Biol Chem 2010, 285(5):3439-3450.
[2].    Vaseva AV, Marchenko ND, Moll UM: The transcription-independent mitochondrial p53 program is a major contributor to nutlin-induced apoptosis in tumor cells. Cell Cycle 2009, 8(11):1711-1719.