Given that the proto-oncogene c-Myc binding E boxes (CACGTG) match the hypoxia response element consensus sequence (A/GCGT), which results in a similar pattern of gene regulation between HIFs and c-Myc, and that the promoter also contains putative c-Myc binding E boxes in its ?340 bp region, we reasonably speculated that might be a target gene of c-Myc. detergent (sodium deoxych 3?mg/mL with Tween 40) was added to lysis buffer B according to the ratio of 1 1:10, and then the mixture was vortexed 2C3 times to wash the nuclei sufficiently. The tube was incubated on ice for 5?min before centrifuging at 1000at 4?C for 3?min. Then the supernatant was discarded, and washing was repeated for 5C10 times. An appropriate amount of 1% NP40 lysate buffer was added to the nucleus and put it in liquid nitrogen for 1?min, then took it out and thawed on ice. Repeated the freezing and thawing process twice, and then vortex vigorously before centrifuging at 16,363together with the cytoplasmic components at 4 C for 30?min. 2.7. GST pull down assay GST and Tacalcitol monohydrate GST-WSB1 expressed in BL21 were purified according to previous report26 and the purified protein was used freshly. The whole cell lysate of 293FT and GST or GST-WSB1 protein was incubated in 1% NP-40 buffer (25?mmol/L Tris, 150?mmol/L NaCl, 10% glycerol and 1% Nonidet P-40, pH?=?8.0) with protease inhibitor cocktails at 4?C for 4?h, and the glutathione-agarose beads were subsequently added to incubate for another 2?h. The beads were washed for 4 times with washing buffer (25?mmol/L Tris, 300?mmol/L NaCl, 10% glycerol and 0.2% Nonidet P-40, pH?=?8.0) and the proteins were resolved by heating at 95?C with loading buffer (25?mmol/L TrisCHCl, 2% SDS, 1?mg/mL bromophenol blue, 50% glycerol and 5% forward: ACTGTGGAGATATAGTCTGGAGTCT; reverse: GTAGCAAGAAGTAGCTGATCTTGTC; forward: GTTCAGTTGCTTGTTCGTGC; reverse: GTTGTGAACATCCCGAGCTAG; forward: GGACCCGCTTCTCTGAAAG; reverse: GTCGAGGTCATAGTTCCTGTTG; forward: GCGACCACAAGGCCCTCAGTACCTC; reverse: AATGTGGTGACAGCCTTGGTGTTGG; forward: CTGGAGAGAGCTGTGAGCGACCGG; reverse: GAGCAGGCCTGGGTCTTGGGTTCG; Fluc values were detected in the Microplate reader. Stop & Glo reagent was used to stop the reaction and start luciferase reaction at the same time, the value of which was used as an internal control. pCDH-WSB1 and pGL4.14-c-Myc promoter-luciferase plasmids as well as pCDH-WSB1 and pGL4.14-TCF/LEF1-luciferase plasmids were transfected on H460 and tested with the luciferase reporter kit (Promega) in the same way. Stop & Glo reagent was used to stop the reaction and start Tacalcitol monohydrate luciferase reaction at the same time, the value of which was used as an internal control. 2.12. Cell proliferation assay Bel-7402 stably overexpressing WSB1 or WSB1 with c-Myc and H460?cells knocking down WSB1 were seeded in 6-well plate with density of 10% respectively. After 2 weeks, cells were stained by the sulforhodamine B and photographed by E-Gel Imager (Bio-Rad). The cloning numbers in each group were counted to calculate the inhibition rate. 2.13. Sphere formation assay The medium of sphere assay was DMEM/F-12 accompanied with penicillin/streptomycin antibiotic, basic Tacalcitol monohydrate fibroblast growth factor (bFGF, 10?ng/mL), human recombinant epidermal growth factor (EGF, 10?ng/mL) and N-2 supplements (1?). Bel-7402?cells and HuH-7? Tacalcitol monohydrate cells were collected and washed with DMEM/F-12 medium to remove the serum, and the cells were counted to reseed with the density of 3000?cells per 2?mL medium per well of the ultra-low attachment 6-well Rabbit polyclonal to DPF1 plate. The plate was incubated at 37?C with 5% CO2 for about 5C8 days when the cells formed and reached the size of 100?m. Photographs were taken by upright microscope (Olympus). 2.14. Immunofluorescence assay Cells were seeded in confocal plates and fixed with 4% paraformaldehyde for about 15?min. The fixed cells were blocked?with?1% BSA for 30?min at room temperature and incubated with?is the width and is the length. The animal experiment was approved by the Institutional Animal Care and Use Committee in Zhejiang University, and was carried out following the institutional guidelines. The IACUC number of the animal experiment was IACUC-s21-027. 2.16. Immunohistochemistry (IHC) HCC tissue array (BC03119b) was purchased and processed from Alena Biotechnology (Xi’an, China) and photographs Tacalcitol monohydrate were taken by upright microscope (Olympus). And the informed written consent from all participants or next of kin was obtained prior to the research. The nude mice were sacrificed at the end of the xenograft experiment after the tumor dissected. The tissues were fixed with 4% paraformaldehyde. Paraffin sections were prepared and the antigen retrieval was applied with EDTA solution (50?mmol/L Tris, 10?mmol/L EDTA, pH?=?9.0) after dewaxing treatment. The tissues were incubated with WSB1 antibody (1:200) and c-Myc antibody (1: 200) at 4?C overnight. DAB chromogenic kit was used to react with the second antibody. Photographs were taken by upright microscope (Olympus). The evaluation standard of staining intensity is as follows: 0 point for no staining, 1 point for yellow, 2 points for.