Fig. 3

Combination treatment reduces the proportion of CD44⁺ and ALDH⁺ OCSC cells. a Sorted Hes1GFP⁺-CD44^+/− /Hes1GFP⁺-ALDH^+/− cells were cultured in CSC medium (96-well plate), and were treated with cisplatin (10 μM), eugenol (2 μM) and the combination of both drugs for 5-days. The viability of spheres was determined by the WST-1 assay. b Sorted Hes1GFP⁺CD44⁺ and Hes1GFP⁺ALDH⁺ cells were treated with vehicle (control), cisplatin, eugenol and combination of both agents for 72 h, trypsinized and cultured as spheres (5000 cells/well) in CSC medium for 10 days. Sphere forming ability was monitored routinely under the microscope and counted and recorded the number of spheres from each group. Data are presented as “cumulative event” and presented as survival stair graph. c Cells were treated with DMSO (control), cisplatin (10 μM; 72 h), eugenol (2 μM; 72 h) or the combination of both drugs (cisplatin 24 h, followed by eugenol for additional 48 h; total 72 h). CD44-FITC antibody was used to sort cells using the NovoCyte flow cytometer. d The ALDEFLOUR assay was performed to identify the CSC populations following treatment as in (c). ALDH specific inhibitor DEAB was used as negative control, ALDH⁺ cells are shown in cells residing in framed area analyzed using NovoExpress software