Sonicator

The generation of soluble chromatin was described previously (9 (link)). The cultures were cross-linked with 1% formaldehyde for 15 min at room temperature, mixing occasionally. The cells were then collected with centrifugation and washed. The cells were again collected with centrifugation, transferred to a microcentrifuge tube and collected with centrifugation. The cell pellet was flash-frozen in liquid nitrogen and stored at −80°C. The cell pellets were resuspended in 400 μl sFA-140 lysis buffer [sFA-140 refers to the FA-140 buffer used for Saccharomyces cerevisiae; 50 mM HEPES-KOH, pH 7.5; 140 mM NaCl; 1 mM ethylenediaminetetraacetic acid (EDTA); 0.1% Triton X-100 and 0.1% sodium deoxycholate]. The cells were lysed by bead-beating with glass beads (BioSpec 11079105). The lysate was separated from the beads and sonicated (Misonix sonicator: Power 6, 10 s, six times with 10-s rest) to shear the chromatin. Directly after sonication, the lysate was placed on dry ice for 10 s. The lysates were centrifuged for 5 min at 4°C at top speed and then for 10 min at 4°C at top speed to separate soluble and insoluble chromatin (∼400 μl). Twenty five microliters (6.25%) of soluble chromatin was removed and processed for DNA input using a PCR purification kit (Qiagen 28106). The remaining soluble chromatin can be immediately used for the immunoprecipitation or it can be stored at −20°C.

Bacterial biomass from the stationary phase of growth (the samples were withdrawn from 120-h-old cultures) was separated from culture media by centrifugation at 4,000×g. It was homogenized (Misonix Sonicator) with 10 ml of a CHCl₃–MeOH mixture (2:1, v/v). A total of 30 µl of IS solution was poured into each sample before extraction. To the crushed cells 2 ml of 0.8 % NaCl was added, the vials were vortexed for 1 min and centrifuged. The lower organic phase was collected, treated with anhydrous sodium sulphate, and evaporated under reduced pressure. The residue was then re-dissolved in 2 ml of methanol/CHCl₃ (4:1, v/v) and stored at −20 °C pending analysis.

The assay was performed by the method with modifications, as described previously [31 (link)]. bEnd.3 cells were seeded onto culture dishes (10-cm). The cells were starved for 24 h in DMEM/F-12 medium without FBS when they reached 90% confluence. After incubation with 10 μM 15d-PGJ₂ for the time intervals, the cells were washed once with ice-cold PBS and scraped into a 1.5 mL tube with 1 mL of PBS added to each dish. Cells were collected by centrifuge at 8000 rpm for 5 min. The pellets were suspended with 300 μL Cytoplasmic Extraction Reagent I (CREI). The suspension was broken by syringes. After being put on ice for 30 min, the lysates were centrifuged at 8000 rpm for 10 min. The pellet was collected as the nucleus fraction and the supernatant as a cytosol fraction. The pellets were resuspended and then sonicated for 5 s twice using a sonicator (Misonix, Farmingdale, NY, USA). The protein concentration of each sample was measured. Samples from these fractions (200 μL protein) were denatured and subjected to SDS-PAGE using a 12% (w/v) running gel. The levels of translocation were identified and quantified by Western blot analysis.

Cells were pelleted and resuspended in 500 μl of PBS containing 20 mM sodium butyrate. Cells were fixed at room temperature with 1% formaldehyde for 8 minutes, and then quenched with 1.25 M glycine. Cells were sonicated using Misonix sonicator under the following conditions to obtain chromatin fragments between 200–700 bp size: Hepatocytes– 65 Amplitude (4 pulses with 1 minute interval between each pulse which was of 16 seconds with 1 second on and 1 second off); Lin^- BM cells– 40 Amplitude (1 pulse of 10 minutes with 30 seconds on and 30 seconds off). ChIP was performed using Low cell ChIP kit from Diagenode (Seraing, Belgium) according to the manufacturer’s protocol using the specific antibodies. Antibodies used for ChIP (anti-JMJD3, anti-EZH2, anti-H3K4me3, anti-H3K9Ac, anti-H3K9me3, anti-H3K27me3, rabbit IgG) were procured from Abcam (Cambridge, UK). Analysis of ChIP samples was carried out by real-time qPCR using the primers (S2 Table) and the efficiency of chromatin immuno-precipitation was calculated as percentage of input (ChIP/Total input) = 2^{[(Ct(1% input)–log₂ (dilution factor))—Ct(ChIP)]} x 100%.

After treatment, harvested cells were sonicated for 5 s at output 1.5 with a sonicator (Misonix, Farmingdale, NY, United States). Then, the cell lysates were centrifuged at 8,000 rpm for 15 min at 4°C. Both the pellet and supernatant were collected, respectively. The pellet was saved as the nuclear fraction. The supernatant was saved for further centrifugation at 14,000 rpm for 60 min at 4°C. The pellet and the supernatant were respectively collected as membrane fraction and cytosolic fraction.

3T3-L1 adipocytes were scraped into a lysis buffer containing 40 mM HEPES (pH 7.4), 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 1.5 mM Na₃VO₄, 10 mM β–glycerophosphate, and 1% Triton X-100, supplemented with EDTA-free phosphatase and a protease inhibitor cocktail (#78441; Thermo Scientific, Waltham, MA, USA). Next, the cells were lysed by sonication twice for 15 sec on ice using a sonicator (Misonix, Farmingdale, NY, USA). After centrifugation at 10,000 × g for 20 min at 4°C, the supernatants were boiled in sodium dodecyl sulfate (SDS) loading buffer at 90°C for 5 min, after which they were loaded (20 μg protein per lane) onto 8% or 12% Tris-glycine gels. Following electrophoresis, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked using 5% skim milk in TBST buffer (20 mM Tris, pH 8.0, 125 m NaCl, 0.5% Tween 20) for 1 h. The blocked membranes were incubated with primary antibodies in TBST overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. Finally, the membranes were visualized using a chemiluminescence reagent (Amersham Bioscience, Piscataway, NJ, USA).