br Western blot assay br For
4.4. Western blot assay
For Western blot analysis, Cycloheximide were treated with the indicated concentrations of drugs for 24 and 48 h and then harvested by scraping
with lysis buffer (1 mM EGTA, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM PMSF, 1 mM Na3VO4, 1 μg/ mL leupeptin, 1 μg/mL aprotinin, 5 mM NaF in 20 mM Tris–HCl buffer, pH 7.5). Cell lysates were centrifuged at 13,000g for 30 min. Total protein was determined and equal amounts of protein were separated by SDS–PAGE and immunoblotted with specific antibodies. Proteins were visualized with enhanced chemiluminescence (Amersham, Buckinghamshire, UK).
4.5. Cell cycle analysis
PC3 cells were seeded on 6-well plates (2.5 × 105/well) and treated with the indicated drugs and concentrations for 24 and 48 h. Then the cells were harvested and trypsinized, washed with PBS (phosphate-buffered saline) and fixed in cold 70% EtOH for at least 30 min at 4 °C. After removal of EtOH by spinning at 2000 rpm for 30 min in a cen-trifuge, the pellets were resuspended in DNA extract buffer (0.2 M Na2HPO4, 0.1 M citric acid; pH 7.8) for 20 min. Then the cells were washed with PBS and stained with propidium iodide (PI, 100 µg/ml RNaseA, 80 µg/ml propidium iodide, 0.1% Triton X-100 in PBS). Cell cycle distribution was measured by BD Accuri™ and C6 Software (BD Biosciences, Franklin Lakes, NJ, USA).
4.6. Molecular modeling studies
The crystal structure of HSP90 (PDB ID: 5GGZ) was downloaded from the Protein Data Bank  The drug design platform, Lead IT  was used for docking analysis. The Lead IT software was also used to prepare the target protein. The target protein was prepared by re-moving the co-crystal ligand and water molecules. Compounds were
protonated in aqueous solution. The binding site was defined as a radius of 10 Å using the co-crystallized ligand as a reference. A hybrid (en-
thalpy and entropy) docking strategy was used. All scoring parameters were used with the default settings.
4.7. Mice xenograft model
Adult (5–6 weeks old) nude male mice were purchased from National Laboratory Animal Center (NLAC) of Taiwan. During experi-mental period, the mice were housed at the Laboratory Animal Center of Taipei Medical University, on a 12-hour light cycle at 21–23 °C and 60–85% humidity. The cancer cells were harvested and resuspended in PBS at 5 × 107 cells/mL. Each mouse was injected s.c. in the right flank with 1 × 107 cells (0.2 mL cell suspension). The tumor size and body weight were monitored twice weekly until their volumes approached 1,200 mm3. On D1 of the study the animals were sorted into treatment groups with tumor sizes of 60–150 mm3. Tumor size, in mm3, was calculated as: Tumor Volume = w2 × l/2 where w = width and l = length in mm of the tumor. All treatment doses were administered at a volume of 10 mL/kg (0.2 mL/20 g mouse), scaled to the body weight of each animal. Control mice receive vehicle treatment (1% CMC and 0.5% Tween80 in ddH2O). All experiments were maintained in accordance with the Institutional Animal Care and Use Committee procedures and guidelines.
Appendix A. Supplementary material
Y. Shen, 17-DMCHAG, a new geldanamycin derivative, inhibits prostate cancer cells through Hsp90 inhibition and survivin downregulation, Cancer Lett. 362 (2015) 83–96;
V.M. Vukovic, R. Bradley, M.D. Karol, Y. Chen, W. Guo, T. Inoue, L.S. Rosen, A first in human, safety, pharmacokinetics, and clinical activity phase I study of once weekly administration of the Hsp90 inhibitor ganetespib (STA-9090) in patients with solid malignancies, BMC Cancer 13 (2013) 152;
 S. Chatterjee, E.H. Huang, I. Christie, B.F. Kurland, T.F. Burns, Acquired resistance to the Hsp90 inhibitor, ganetespib, in KRAS-mutant NSCLC is mediated via re-activation of the ERK-p90RSK-mTOR signaling network, Mol. Cancer Ther. 16 Bioorganic Chemistry 91 (2019) 103119
C. Hudis, J. O'Shaughnessy, J.A. Baselga, A multicenter trial evaluating re-taspimycin HCL (IPI-504) plus trastuzumab in patients with advanced or metastatic HER2-positive breast cancer, Breast Cancer Res. Treat. 139 (2013) 107–113.
AMPK Promotes SPOP-Mediated NANOG Degradation to Regulate Prostate Cancer Cell Stemness
Graphical Abstract Authors
Xinbo Wang, Jiali Jin,
Wang et al. uncover a regulation mechanism by which NANOG stability, which is involved in the regulation of the stem-like characteristics of prostate cancer cells, is dictated by SPOP-mediated degradation and is controlled by AMPK-BRAF-mediated phosphorylation of NANOG at Ser68.