Ten fields were randomly taken to count the number of cells. concentration of 10 M and stored at -20C. For studies, APG-1387 was dissolved in 10% PEG400/5% EL/85% PBS. SP cells analysis The S18 or S26 cells were untreated or treated with the compounds to test for 24 h, then harvested and resuspended at 106 cells/mL density in ice-cold DMEM (supplemented with 2% fetal bovine serum). The DNA binding dye hoechst 33342 (Sigma-Aldrich) was added at a final concentration of 5 g/mL and incubated for 90 min at 37C in the dark with interval mixing. As a negative control, a subset of the cells were incubated with 5 M fumitremorgin C (FTC, an inhibitor of ABCG2 that could block the pumping out of hoechst 33342 in CSCs, Merck) for 5 min prior to hoechst 33342 dyeing. After hoechst staining, cells were washed twice then pelleted and maintained at 4C before FACS analysis. FACS analysis was performed on COULTER EPICS ALTRA? Flow Cytometer (Beckman Coulter). The hoechst dye was excited with UV laser at 350 nm and its fluorescence Levamlodipine besylate was measured at two wavelengths using a 450/40 BP filter (hoechst Blue) and a 675 long pass filter (hoechst Red). Flow cytometry data were analyzed using FlowJo software. At least three impartial experiments were performed. CD44 cells analysis 1106 S18 or S26 cells were suspended in 100 L PBS for CD44-PE or IgG-PE antibody labeling. CD44-PE antibody (clone: DB105) and unfavorable control IgG-PE antibody were obtained from Miltenyi Biotec GmbH (Germany) and used to label cells following the manufacturer’s instructions. Flow cytometric analysis was performed using a Beckman Coulter filtered with a 488 nm laser. At least three impartial experiments were performed. Quantitative real-time PCR Total RNA of S18 or S26 cell was extracted using trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized using High Capacity RNA-to-cDNA Kit (Applied BiosystemsTM) according to the manufacturer’s instructions. Real-time PCR amplification was performed by SYBR? Green PCR Grasp Mix (Applied BiosystemsTM) on a Hard-Shell PCR Plates (Bio-Rad). Relative quantification of each target gene was normalized by using an endogenous control (GAPDH). qPCR and analyses were performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad). Cell proliferation and cytotoxicity assay Cell proliferation and cytotoxicity was measured using Cell Counting Kit-8 (Dojindo). S18 and S26 cells were counted and plated in triplicate at 2000-3000 cells per well (200 L) in 96-well plates (Falcon), and allowed to adhere overnight. For individual groups, compounds (cisplatin, 5-fluorouracil, APG-1387) were added to the wells in concentration gradients. Cell viability was measured 72 h later by adding 10 L CCK8 per well and incubated 1-4h. The observation value was detected at 450 nm, Prism software was used to calculate the IC50. All experiments were performed in 6 replicates per trial, with three impartial trials in total and the average percentages of cell viability are shown. Colony formation assay S18 or S26 cells were Levamlodipine besylate plated in HD3 triplicate at 100 cells per well in 6-well plates (Falcon), and cultured in DMEM (supplemented with 10% fetal bovine serum) for 7-10 Levamlodipine besylate days. Then, the cells were washed twice with PBS and fixed in methanol for 10 min. After washing with PBS twice, the Levamlodipine besylate cells were dyed with crystal violet for 30 min. Then, the crystal violet was washed out and the number of colonies was counted. Images are shown as representatives of three impartial experiments. Sphere formation assay S18 or S26 cells were plated in triplicate at 1000 cells per well in ultra-low attachment 6-well plates (Corning), and cultured in DMEM/F12 medium (Invitrogen) with 20 ng/mL recombinant human basic Levamlodipine besylate fibroblast growth factor (Invitrogen), 20 ng/mL recombinant human epidermal growth factor (BD Biosciences), B-27 supplement (Invitrogen) and compounds to be tested for ~2 weeks. The spheres were counted under a light microscope. Images are shown as representatives of three impartial experiments. migration assay S18 or S26 cells were suspended in serum-free.