Nuclei ez prep nuclei isolation kit

A Nuclei EZ prep nuclei isolation kit (Sigma-Aldrich, MO, USA) was used to isolate nuclei and count only nuclear DNA in order to quantify the amount of live cells after the different culture conditions. Islets were centrifuged at 500 G for 5 min. The supernatant was removed and the cells were washed with PBS the supernatant was discarded and 1 ml of cold Nuclei EZ prep nuclei isolation lysis buffer was added. Cells were vortexed and incubated at 4 °C for 5 min. The remaining cellular material was centrifuged at 500 G for 5 min to sediment nuclei and supernatant with other cellular components was discarded. Isolated nuclei were subsequently used to quantify the DNA content.
Quantification of DNA in islet nuclei was performed using a Quant-iT™ PicoGreen® dsDNA Assay Kit (Live Technologies, The Netherlands)46 (link). The nuclei were incubated with 100 μl of TE buffer (200 mM Tris-HCl, 20 mM EDTA, pH 7.5). After adding the solution nuclei were sonicated for 10 sec at 30% power and plated in a 96 well-plate. Reagent for fluorescent detection was prepared by making a 200-fold dilution of the Quanti-iT PicoGreen reagent in TE buffer and addition of 100 μl to the samples. Quantification of dsDNA was done in a fluorescence microplate reader Thermo Scientific Varioskan® (Thermo Scientific, MO, USA) at excitation and emission wavelengths of 480 and 520 nm respectively.

Starved HMVEC-d cells were infected with KSHV (20 DNA copies/cell) for 2 hr, washed with trypsin-EDTA, and subjected to nuclear isolation using a Nuclei EZ Prep Nuclei Isolation Kit (Sigma-Aldrich) as previously described [26] (link). Briefly, cells were lysed with mild lysis buffer and the nuclei were pelleted by centrifugation at 500×g for 5 minutes. The cytoplasmic contaminants that were loosely attached to the nuclei were removed by repeated washing with the mild lysis buffer. The DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen). Equal amounts of DNA were quantified with ORF73-specific primers by real-time DNA PCR. The absolute copy number was determined using an ORF73 standard curve as described in the entry experiment.

To determine the cellular localization of CASC9, nuclear fraction was isolated from cytoplasm according to the manufacturer’s instructions for NUCLEI EZ PREP NUCLEI ISOLATION KIT (Sigma, USA). Firstly, the cells were washed gently with ice-cold PBS twice. Then 1 ml ice cold Lysis Solution was added to the the 25cm² flask and about 3 × 10⁶cells were harvested with a cell scraper on ice. The cell lysate was incubated on ice for 5 min. After centrifuging at 500 g for 5 min at 4 °C, the precipitate containing the nuclear RNA was isolated from the supernatant containing the cytoplasmic RNA. Finally, the supernatant was carefully removed to a new 1.5 ml EP tube. The precipitate was washed by PBS twice and resuspended by Nuclei EZ storage buffer.

Cells were collected and lysed on ice in lysis buffer containing 0.5% Triton X-100, 20 mM HEPES pH 7.4, 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl₂, 10 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM EGTA, 20 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride for 20 min, followed by centrifugation at 12,000 r.p.m. for 15 min to extract clear lysates. For immunoprecipitation, the cell lysates were incubated with 1 μg of antibody and A-Sepharose beads at 4 °C overnight. After incubation, the beads were washed four times with lysis buffer, and the precipitates were eluted with 2 × sample buffer. The eluates and whole-cell extracts were resolved by SDS–PAGE followed by immunoblotting with antibodies. Nuclear fractionation was performed using the NUCLEI EZ PREP kit purchased from Sigma in accordance with the manufacturer's instructions. For cell fractionation, we used the NUCLEI EZ PREP NUCLEI ISOLATION KIT from Sigma (Cat No. NUC-101) to isolate nuclear and cytoplasmic proteins. Densitometric quantification of the western blot results was performed on images of scanned films using ImageJ software. All uncropped western blots can be found in Supplementary Fig. 9.

Cells at a density of 20.0×10⁴ cells/well were seeded into 6-well plates and treated with SB (50 µM) for 48 h at a temperature of 37°C. DMSO was used as a negative control, the concentration of DMSO was 1‰. The cytoplasmic and nuclear proteins were separated and extracted using a Nuclei EZ Prep Nuclei Isolation kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. The expression levels of the different proteins were analyzed using western blotting.