The West Nile virus capsid protein blocks apoptosis through a phosphatidylinositol 3-kinase-dependent mechanism. cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3. CONCLUSIONS The results suggest that GSK3 participates at the late stages of the DENV replication cycle, where viral activation may promote 11-oxo-mogroside V apoptosis and release of viral particles. – Viruses were cultured in C6/36 HT (high temperature) cells from – GSK3 small molecule inhibitor Kin-001-184 was donated by Dr Priscilla Yang (Harvard Medical School). CT 99021 (Kin-001-157) was obtained from Axon (cat # 1386 Groningen – 11-oxo-mogroside V The Netherlands). Mycophenolic acid (MPA), obtained from Sigma-Aldrich (Ref. M3536-250G), was used as positive control for the inhibition of DENV replication. GSK3 inhibitors were dissolved in dimethyl sulfoxide (DMSO, Sigma) and MPA was dissolved in methanol (50 mg/mL). The primary antibodies used were rabbit -GSK3 (cat # 9369), rabbit -phospho-GSK3-Ser9 (cat # 9323), rabbit -Akt (cat # 9272), rabbit -phospho-Akt-Ser473 (cat # 9271S), rabbit -GADPH (cat # 2118), and rabbit –catenin (cat # 9587) (Cell Signalling, Danvers, MA). For immunofluorescence, secondary antibodies conjugated to fluorophores Alexa 488 and Alexa 594 (Molecular Probes, Eugene, OR) were used, and Hoechst 33258 (Thermo Fisher Scientific, cat # H3569) was used for nuclear labelling. The secondary antibodies used were IRDye 800CW goat anti-mouse and IRDye 680 goat anti-rabbit (1:15000) (Li-COR, Lincoln, NE). Protein quantification was performed using BCA Protein Assay kit (Pierce, Rabbit polyclonal to ZNF624.Zinc-finger proteins contain DNA-binding domains and have a wide variety of functions, mostof which encompass some form of transcriptional activation or repression. The majority ofzinc-finger proteins contain a Krppel-type DNA binding domain and a KRAB domain, which isthought to interact with KAP1, thereby recruiting histone modifying proteins. Zinc finger protein624 (ZNF624) is a 739 amino acid member of the Krppel C2H2-type zinc-finger protein family.Localized to the nucleus, ZNF624 contains 21 C2H2-type zinc fingers through which it is thought tobe involved in DNA-binding and transcriptional regulation Thermo Scientific ref 23225). – Following treatments with inhibitors, the viability of Huh7 cells was tested using the MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium bromide) assay. Cells were seeded onto 96-well plates and incubated for 24 h. The culture medium was replaced with DMEM-containing GSK3 or MPA inhibitors at concentrations of 5, 10, 20, and 40 M, prepared by serial dilution. After 24 h incubation, the medium was replaced with 50 L of MTT [0.5 mg/mL in phosphate-buffered saline (PBS)], followed by 11-oxo-mogroside V 3 h of incubation at 37oC. DMSO (100 L) was added to solubilise formazan crystals and incubated for 15 min. Absorbance at 450 nm measured using a microplate reader (Benchmark, Bio-Rad Laboratories, Hercules, CA, USA). Three independent experiments were performed with each treatment in triplicates. – The prototype strain DENV-2 New Guinea C (NGC) donated by Maria Elena Pe?aranda and Eva Harris (Sustainable Sciences Institute and the University of California) was used in all infection experiments. Virus stocks were used for infection of C6/36 HT cells at low multiplicity of infection (MOI) (0.01 PFU/cell). Once infected, cells were incubated for seven days and supernatants were aliquoted and stored at -80oC until titration. Viral titre 11-oxo-mogroside V determination was performed by diluting virus (10-1-10-5) in serum-free medium. Vero cell monolayers grown to 90% confluence in 48-well plates were inoculated with diluted virus. After 1 h adsorption at 37oC, viral inoculum was removed. Cells were washed with PBS and covered with 2% carboxymethyl cellulose (medium viscosity carboxymethyl cellulose, Sigma-Aldrich) in DMEM containing 2% foetal bovine serum (FBS). After seven days of incubation, cells were fixed with 4% paraformaldehyde and stained with 0.5% violet crystal prepared in 20% methanol. Viral titre calculations were done by counting two replica plates from three independent experiments (n = 6). – Huh7 cells (2 105) were seeded onto 6-well plates for.
