*4.5. The Soft Agar Colony Formation Assay*

JB6 Cl41 cells (2.4 <sup>×</sup> <sup>10</sup>4) were exposed to EGF (1 ng/mL) and treated with rHct-S3 (1, 2, and 4 <sup>μ</sup>M) in 1 mL of 0.3% Basal Medium Eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 μg/mL gentamicin. The cultures were maintained at 37 ◦C in a 5% CO2 incubator for 14 days, and the cell's colonies were scored using a microscope, Motic AE 20 (XiangAn, Xiamen, China) and the ImageJ software.

To estimate the effect of rHct-S3 on colony formation, human cancer cells (2.4 <sup>×</sup> 104 /mL) were seeded into 6-well plate and treated with rHct-S3 (1, 2, and 4 μM) or cisplatin (3 μM) in 1 mL of 0.3% Basal Medium Eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 μg/mL gentamicin. The cultures were maintained in a 37 ◦C, 5% CO2 incubator for 14 days, and the cell colonies were scored as described above.
