The IC50 value for the DU-145 cells was 0.3 M, whereas for 22Rv1 and LNCaP it was 1.2 and 4 M, respectively. plasmid using the FugeneHD transfection reagent (Promega). Individual hygromycin B-resistant cell colonies were isolated, expanded, and screened for the DHT-inducible expression of luciferase. To assess the AR transactivation activity following 24 h drug treatment, cells were lysed, and the level of firefly luciferase was measured using the Luciferase Assay System (Promega), according to the manufacturers instructions. 2.5. Transient Transfection with the ARE-Luc Vector LNCaP cells were seeded on a 96-well plate (10,000 cells per well) overnight, co-transfected with the (AREIII)3-(AREII)3-(AREI)3-tk-Luc plasmid or the pG4.14luc/hygro (Promega) and pCMV-SEAP plasmid (a kind gift from Dr. S. Schlatter) using the FugeneHD transfection reagent. Cells were treated with an experimental compound 24 h after transfection, PSC-833 (Valspodar) and reporter gene activity was assayed after a further 24 h. Luciferase activities were normalized to the corresponding SEAP activity, which was used as the transfection efficiency control. 2.6. Cell Viability Assays Cell viability was measured using an MTT (3-(4,5-diethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, cells (2 105) were seeded in 24-well plates, left to attach overnight, and treated with the drugs for 72 h. After exposure, 0.5 mg/mL MTT was added into each well, and the plates were incubated for 4 h at 37 C. The medium was removed, and the remaining formazan crystals were dissolved in DMSO. Absorbance was measured at 540 nm. The viability of the cells in the 3D spheroid cultures was measured using a resazurin assay. Briefly, single spheroids were grown in 200 L of culture medium in 96-well plates. Following the drug treatment, 100 L of the medium was removed, and replaced with a 20 L resazurin solution (0.15 mg/mL) in PBS. After incubation for 4 h at 37 C, the absorbance was measured at an excitation/emission wavelength of 544/590 nm. Cell viability was expressed as the percentage of the vehicle-treated control and the cytotoxic effect of the drug was assessed by the IC50 values. 2.7. Western Blot Analysis Following PSC-833 (Valspodar) the drug treatment, cells were lysed PSC-833 (Valspodar) for 20 min on ice with buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA 1% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 50 mM -glycerolophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Proteins (50 g) were separated by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used: anti-PSA (#5877 Cell Signaling Technology, Danvers, MA, USA), anti-AR (#5153S Cell Signaling Technology), anti-GAPDH-HRP conjugate (#8884S Cell Signaling Technology), anti-H2AX (#05-636-I, Millipore, Burlington, MA, USA), #anti-p53 (DO-I, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p21 (#2947 Cell Signaling Technology), anti-PARP (#9542 Cell Signaling Technology), and anti–actin (C4, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoblots were developed using the Li-COR Odyssey imaging system, except for the anti-PSA, anti-AR, and anti-GAPDH-HRP conjugate, which were visualized using an enhanced chemiluminescence detection kit, the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA). 2.8. ChIP Assay for the Androgen Receptor LNCaP cells were cultured until an 80% confluence and exposed to C-1311. Proteins were cross-linked with formaldehyde, and then the cells were harvested, lysed, and the DNA sonicated with a VCX-130 sonicator (Sonics & Materials Inc., Newtown, CT, USA). Chromatin immunoprecipitation was performed using the Magna ChIP? A/G Chromatin Immunoprecipitation Kit (EMD Millipore, Burlington, MA, USA) according to the manufacturers protocol. The following antibodies were used: control mouse IgG (EMD Millipore, Burlington, MA, USA) and anti-androgen receptor antibodyChIP Grade (#ab74272, Abcam, City, UK). The relative enrichment of the gene sequences was analyzed with real-time PCR using the SYBR Green I Master Mix on a LightCycler 96 (Roche, Basel, Switzerland). Primers P1 complementary to the were as follows: forward 5-GAGTGCTGGTGTCTTAGGGC-3 and reverse 5-GCTAGCACTTGCTGTTCTGC-3; primers P2 complementary to the PSA CSF3R intron 1 were as follows: forward 5-CCTCTTCCAGCAACTGAACC-3 and reverse 5-TCAGGGTTGACAGGAGGAAC-3. The AR negative-binding region in the first intron of the PSA was identified using Matinspector software . The amplification of the soluble chromatin prior to immunoprecipitation was PSC-833 (Valspodar) used as an PSC-833 (Valspodar) input control. Quantification was performed using the dCt method with the Ct obtained for input DNA as a reference value: 1000 2-dCt, where dCt =.
- Genes Analyzed We analyzed the expression level of genes previously reported as related to the following processes or pathways: JAs biosynthesis (and [33,70]), JA signaling (and ), MBW complex (and repressor ), and anthocyanin and PA biosynthetic genes ([15,71,72], Supplementary Table S10)
- The PAR-6 polarity protein regulates dendritic spine morphogenesis through p190 RhoGAP and the Rho GTPase
- Niclosamide blocks the acidification of endosomes, albeit utilizing a different system that’s not yet elucidated 166 fully, 167
- Marked are (reddish colored) and (blue)