4T1 cells with a density of 2 × 105 cells/well were grown into the six-well plate and incubated until confluent 80%. The cells were then treated with selected concentrations of piperine and PGV-1. After being incubated for 24 h, the cells were rinsed with PBS two times, and the May-Grünwald-Giemsa stain (0.25% w/v, in methanol) (Sigma-USA) was added to each well up to 5 min. After that, the cells were flushed with phosphate buffer pH 7.2 for 1.5 min, and a Giemsa stain diluted with phosphate buffer (1: 50) was added to the well. After waiting for about 15 to 20 min at room temperature, the cells were rinsed briefly by deionized water and finally air-dried. Observations were made using an inverted microscope (Leica) Mitotic catastrophe analysis is indicated by cells with micronucleus or polynuclear (3 (link), 11 (link)). The percentage of mitotic catastrophe cells was calculated by two people with blinded observation method.