33377-72-9 br Tumorsphere culture br Cells transfected by co
2.7. Tumorsphere culture
Cells transfected by control or specific shRNAs were digested into single cell with trypsin-EDTA and were respectively seeded in 35 mm non-treated cell culture dishes (BIOFIL, 2000 cells/dish) with con-tinuous culture in DMEM/F12 medium (HyClone) containing B27 supplement (gibco), N2 supplement (gibco), bFGF (20 ng/ml), and EGF (20 ng/ml) for two weeks. Then the picture of the formed tumorspheres were taken by inverted microscope（Leica）and spheres with dia-meters larger than 50 µm were counted.
2.8. Plasmids and lentiviral infections
BPTF-targeting shRNAs (shBPTF-1, shBPTF-2, shBPTF-3) and con-trol non-targeting shRNA plasmids purchased from GeneCopoeia were
used for RNA interference. The infection was mediated by lentivirus and conducted according to the manufacturer's protocol (Lenti-Pac HIV expression packaging kit, GeneCopoeia). Briefly, one 10-cm culture dish containing 1.3–1.5 × 106 293 T 33377-72-9 were transfected with 2.5 μg shRNA expression plasmids and 5.0 μl lenti-Pac HIV mix using Lipofectamine 3000 transfection reagent (Invitrogen). Supernatants were collected after 48 h, centrifuged and filtered, and the viral titre was determined by serial dilutions. Then the virus was used to infect target cells to obtain the stably transduced cells after puromycin se-lection.
2.9. Acridine orange/ethidium bromide (AO/EB) fluorescence staining
The HepG2 cells and Bel7402 cells seeded in 6-well plates and transfected with shRNA for 24 h were then treated with cis-Dichlorodiamineplatinum (DDP, 26.7uM/well) for another 24 h. Cells were collected and washed with PBS to remove detached cells. AO (Solarbio, China) and EB (Solarbio, China) were respectively diluted 100 times in PBS and mixed according to 1:1. 250ul mixed liquor was added into each well and discarded after shaking several seconds. The staining results were observed under the inverted fluorescence micro-scope（Leica）using blue light as excitation light.
Detection of cell apoptosis was based on FACS analysis by FITC-AV/ PI staining. Cells were plated in 6-well plates and transfected with the negative control shRNA plasmids or specific shRNA plasmids for 24 h followed by cis-Dichlorodiamineplatinum (DDP, 26.7uM/well) treat-ment for another 24 h. Then the cells were harvested, washed twice in ice-cold PBS with 2% BSA and centrifuged at 300 ×g for 3 min. The cells were resuspended in 500 μl binding buffer and stained with 5ul Annexin V-FITC (AV), 5ul propidiumiodide (PI) using an Annexin V-FITC/PI staining kit (KeyGene BioTech). The status of cell apoptosis was analyzed by flow cytometry (BD ACCURI C6).
Expression of stemness-associated marker, CD24 and CD44, was detected by flow cytometer. Cells were plated in 6-well plates and transfected with siRNA for 10 h. After continuous culture of 48 h, cells were digested with trypsin-EDTA and washed twice in ice-cold PBS containing 2% BSA and centrifuged at 300 ×g for 3 min. Cells were divided into two groups and resuspended in 100 μl PBS with 2% BSA on ice. Then the antibody APC-IgG, PE-IgG and APC-CD44, PE-CD44 (BD Pharmingen) were respectively added into single tube of each group on ice to incubate for 30 min. The fluorescence value was detected finally by flow cytometer.
2.12. Lysate preparation from tissues
The experimental materials used for lysate preparation include lung tissue, xenografts of HCC cells in mice and hepatic carcinoma tumors, adjacent normal tissues obtained from patients who underwent surgery therapy at The First Affiliated Hospital Of Dalian Medical University between 2015 and 2016 with the consent of the patients. These tissues were washed with PBS to remove blood, and transferred to liquid ni-trogen immediately. Tissues were grinded by TGrinder (TIANGEN) into 500ul RIPA buffer with protease inhibitor for 5 min and sonicated for 24 s on ice. Then the lysate were centrifuged at 12,000 ×g for 10 min at 4 ℃, and the supernatants were transferred to new tubes for the fol-lowing determinations.
HepG2 cells with stable knockdown of BPTF were used to perform ChIP assay. First of all, 1 × 107 cells were fixed with 1% formaldehyde for 10 min at RT. Next, 10% 1.25 M glycine was added in the mixture for 5 min to end the excessive crosslink. The mixture was abandoned, the cells were washed three times with cold PBS and then were scraped and harvested in PBS buffer containing protease inhibitors and centrifuged at 240 g for 4 min at 4 ℃. The cell pellets were resuspended twice with PBS buffer containing protease inhibitors and centrifuged at 600 g for 4 min at 4 ℃. The finally collected cells were sonicated five times in IP buffer (SDS buffer: Triton buffer=2:1) for 5 s each time, centrifuged at 14,000 g for 20 min at 4 ℃ and supernatants were transferred to a new tube. 25ul protein A/G agarose beads (Santa Cruz Biotechnology) were mixed with the above supernatant containing 1 mg total proteins and rotated for 30 min at 4 ℃. After centrifugation for 15 min at full speed, the chromatin in the supernatant was immunoprecipitated overnight with 2 μg antibodies against BPTF (Santa Cruz Biotechnology) or IgG (Santa Cruz Biotechnology). Then 25ul protein A/G agarose beads were added into the mixture and rotated for 12 h at 4 ℃. Beads were then washed sequentially for 5 min with the following buffers: 1 ml mixed wash buffer, buffer 500, LiCl/detergent wash buffer twice, and TE buffer one time. The beads were reversely cross-linked by heating at 65 ℃ for 12 h in buffer containing 1% SDS, 0.1 M NaHCO3. After mi-crocentrifuge briefly, the supernatant was treated with 1 ml RNaseA at 37 ℃ for 15 min and digested with 2 ml proteinase K solution at 37 ℃ for 2 h. DNA was finally obtained by LiCl, phenol/chloroform and ethanol extractions and used as DNA templates to amplify the specific hTERT promoter region. The primers used for PCR reaction was as following: forward primer, 5′- TTTCCCACCCTTTCTCGACG-3′; reverse primer, 5′-CAGCGGAGAGAGGTCGAATC −3′