The cells were then fixed with 4% paraformalde hyde for 30 min at room temperature, treated with 2% H2O2 for 30 min followed by 0. 3% Triton X 100 for 30 min, and then stained with the following antibodies anti Oct3/4, anti CD133, anti BCRP, and anti ALDH. The negative control omitted the primary anti body. Ku-0059436 Cells were incubated in a humidified box at 4 C overnight, and then the secondary antibody was added to the cells, followed by incubation at room temperature for 30 min. After the reaction with DAB, the cells were smeared onto slides, examined by microscopy and photographed with a digital camera connected to the microscope. Cell cycle and apoptosis analyses Cell cycle and apoptosis analyses were both performed after 24 hours of sorting.

The cell cycle was examined using a CycleTEST PLUS DNA Reagent Kit follow ing the manufacturers instructions. Cell apoptosis was examined using an Annexin V FITC Apoptosis Detection Kit following the manufac turers instructions. Briefly, the cells were counted and 5 105 1 106 cells of each group were centrifuged at 179 g for 10 min. After the supernatant was removed, cold PBS was added to the cell pellet, followed by gentle vor texing to resuspend the cells. Then, the cells were washed twice, resuspended in 200 uL binding buffer containing 10 uL annexin V FITC, and gently mixed and incubated at room temperature for 15 min while protected from light. Finally, 300 uL binding buffer and 50 uL PI were added to the cell suspension, followed by flow cytometric analysis. Each sample was prepared in triplicate.

In vivo xenografting in immunodeficient mice NOD/SCID mice were purchased from the Animal Insti tute of the Chinese Academy of Medical Science and Peking Union Medical College 2009 0004 and maintained in microisola tor cages. All experiments were approved by the Animal Care Committee of CAMS and PUMC. Freshly sorted SP and non SP cells in 200 ul Matrigel diluted in PBS at a 1 1 ratio were injected subcuta neously into the left axillary fossa of female NOD/SCID mice. Groups of mice were inoculated with SP or non SP cells at 1 105, 1 104 and 2 103 cells, respectively. Tumor appearance was inspected weekly by visual observation and palpation. Mice were sacrificed after 8 weeks and the tumors were harvested, measured, and photographed.

Tumor volumes were mea sured using a digital caliper and approximated according to the formula V 1 2ab2, where a and b are the long and short diameters of the tumor, respectively. Tu mors were fixed in 10% buffered formalin, embedded in paraffin, and then sections were prepared for H E staining. Chemoresistance analysis The sensitivities to chemotherapeutic reagents of HeLa, SP, and non SP cells were assessed using an MTS assay. Briefly, 2 103 cells per well were seeded on 96 well plates in 200 ul per well of appropriate growth Dacomitinib medium. After 24 hours, the cells were treated with TSA at vari ous concentrations.