The human myeloid cells KG1 were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Grand Island, NY) at 37°C in a humidified 5% CO2 atmosphere. In each experiment, logarithmically growing cells were seeded into 5 ml of medium at a density 5 × 105 cells/ml. In the treatment experiments, cells were exposed to the HDACI 4 mM PB or DNMTI 25 μM RG108 the time indicated.
Separation of mononuclear cells from human blood
Mononuclear cells from whole-blood samples from donors were obtained by buffy coat centrifugation from the blood bank (Linkoping University Hospital, Sweden), see also Ethics Statement. The buffy coat (50 ml) was mixed with 1 vol of 0.9% NaCl and 2 vol of 2% dextran in 0.9% NaCl and allowed the fluid separation for 40 min at 4°C. The upper layer was collected, centrifuged at 300 × g for 10 min at 4°C, the pellet suspended in cold Krebs-Ringer-Glucose (KRG) solution without Ca2+ and slowly transferred onto a Lymphoprep (Axis Shield, Oslo, Norway) gradient. After centrifugation at 450 × g for 30 min at 4°C, cells from the mononuclear layer were collected, diluted and washed in cold KRG without Ca2+ by centrifugation at 200 × g for 10 min at 4°C. Pelleted erythrocytes were lysed in cold water for 30 sec following a brief addition of 1:3 vol of 3.4% NaCl and 0.55 vol of KRG without Ca2+. Mononuclear cells were pelleted, resuspended and washed twice in PBS by centrifugation at 220 × g for 10 min at 4°C.
The study was conducted in accordance with the Declaration of Helsinki. Human blood was collected at the blood bank at Linkoping University Hospital by employees at the blood bank division and written consent for research use of donated blood was obtained from all donors. Since blood donation is classified as negligible risk to the donors and since only anonymized samples were delivered to the researchers, the research did not require ethical approval according to paragraph 4 of the Swedish Law (2003: 460; http://www.lagboken.se/dokument/Lagar-och-forordningar/4060/Lag-2003_460-om-etikprovning-av-forskning-som-avser-manniskor?id=64991) on Ethical Conduct in Human Research.
Isolation of CD34+ cells
CD34+ cells were isolated with the CD34 MicroBead Kit according to the manufacturer’s instructions (Miltenyi Biotec, Germany). Briefly, mononuclear cells were diluted with Isolation buffer containing PBS supplemented with 0.5% BSA and 2 mM EDTA (1:2), and cell clumps were removed by filtering through 30 μM nylon mesh (Miltenyi Biotec, Germany). Then cells were counted and resuspended in Isolation buffer for the up to 108 total cells. Cells were labeled by adding FcR Blocking reagent and CD34 MicroBeads for 30 min at 4–8°C. After centrifugation at 200 × g for 10 min at 4°C, cell suspension was applied onto the LS column, unlabelled cell fraction in the effluent was removed and labeled cells were separated using MidiMACs separator. The purity of isolated CD34+ cells was evaluated by flow cytometry and fluorescence microscopy. Flow cytometric analysis by the use of Becton-Dickinson FACS (Becton-Dickinson FACS Calibur, San Jose, CA) demonstrated a purity of >65% CD34+ cells. For the analysis of histones, the nuclear fraction was isolated from 2.5-3 × 106 CD34+ cells; genomic DNA was prepared from about 4 × 107 CD34+ cells.
Isolation of neutrophils from healthy human blood
Defibrinated fresh blood was carefully laid on Polymorphprep™ (Nycomed Pharma AS, Oslo, Norway): Lymphoprep gradient (4:1) and centrifuged in swing-out centrifuge at 600 × g for 45 min at room temperature. The uppermost layers down to the granulocyte band were aspirated, and the very diffuse band with granulocytes (neutrophils) collected and diluted with PBS, pH 7.3. After centrifugation at 600 × g for 10 min at room temperature, erythrocytes from the pellet were removed by lysis in water as described above, the pellet of neutrophils resuspended in PBS.
Histone isolation and analysis
Cells (5 × 106 to 107) were harvested by centrifugation at 500 × g for 6 min, washed twice in ice cold PBS, suspended in Nuclei EZ lysis buffer (Sigma, St. Louis, MO) and nuclei isolated as described by manufacture. For preparation of histones, isolated nuclei were suspended in 5 vol. of 0.4 N H2SO4 by stirring and incubated overnight at 0°C. The supernatant was collected by centrifugation at 15,000 xg for 10 min at +2°C and the sediment was extracted once more. After centrifugation, both extracts were combined and histones were precipitated by adding 5 vol. of ethanol at -20°C overnight. The precipitated histones were collected by centrifugation, washed several times with ethanol and stored at -20°C until analysis.
Histones (5 μg) were dissolved in a buffer containing 0.9 M acetic acid, 10% glycerol, 6.25 M urea and 5% β-mercaptoethanol, and separated on 15% polyacrylamide gel containing 6 M urea and 0.9 M acetic acid by using 0.9 M acetic acid as a buffer . Histones were detected in AUT system (15% polyacrylamide, 6 M urea, 4 mM Triton X-100 and 0.9 M acetic acid) . After electrophoresis, the gel was stained with Brilliant Blue G-colloidal (Sigma, St. Louis, MO) or blots were probed with primary antibodies against total histone H3 (Abcam, Cambridge PLC.) and secondary antibodies, or fractionated in SDS/PAGE system. Immunoreactive bands were detected by enhanced chemiluminescence according to the manufacturer’s instruction (Western Bright ECL, Advansta Corporation, Menlo Park, CA).
Bisulfite modification and methylation-specific PCR
The methylation status of gene promoters was determined with the EZ DNA methylation-Direct™ kit (Zymo Research, Irvine, CA). Briefly, cells (1 - 9 × 105) were digested in the reaction mixture with proteinase K at 50°C for 20 min. Bisulfite conversion of DNA was performed according to the manufacturer’s instruction. Thus after conversion of all unmethylated cytosines to uracils, the modified DNA was purified using a Zymo-Spin™ IC column and used for PCR amplification. The primers, forward (F) or reverse (R), for methylated (M) and unmethylated (U) promoters of the target genes were as follows: E-cadherin (MF) 5′- CAA TTA GCG GTA CGG GGG GC-3′, E-cadherin (MR) 5′-CGA AAA CAA ACG CCG AAT ACG-3′; E-cadherin (UF) 5′-TTA GTT AAT TAG TGG TAT GGG GGG TGG- 3′; E-cadherin (UR) 5′-ACC AAA CAA AAA CAA ACA CCA AAT ACA-3′; p15 (MF), 5′-GCG TTC GTA TTT TGC GGT T-3′; p15 (MR) 5′-CGT ACA ATA ACC GAA CGA CCG A-3′; p15 (UF), 5′-TGT GAT GTG TTT GTA TTT TGT GGT T-3′; p15 (UR), 5′-CCA TAC AAT AAC CAA ACA ACC AA-3′. p16 (MF), 5′-TTA TTA GAG GGT GGG GCG GAT CGC -3′; p16 (MR) 5′-GAC CCC GAA CCG CGA CCG TAA-3′; p16 (UF), 5′-TTA TTA GAG GCT GGG GTG GAT TGT-3′; p16 (UR), 5′-CAA CCC CAA ACC ACA ACC ATA A-3′. RARβ (MF), 5′-GGA TTG GGA TGT CGA GAA C-3′; RARβ (MR) 5′-TAC AAA AAA CCT TCC GAA TAC G-3′; RARβ (UF), 5′-AGG ATT GGG ATG TTG AGA ATG-3′; RARβ (UR), 5′-TTA CAA AAA ACC TTC CAA ATA CA-3′. Cycling conditions: 95°C for 5 min, 40 cycles (95°C for 30 s, annealing temperature 66°C (for E-cadherin and p15 Met), 62°C (for E-cadherin Unmet and p16 Met), 57°C (for p15 and p16 Unmet, RARβ) for 30 s, 72°C for 30 s), 72°C for 10 min. The products were electrophoresed on 3% agarose gel, stained with ethidium bromide, and photographed. The product sizes were as follows: for p15, 150 bp; for E-cadherin, 170 bp, p16, 150 bp, RARβ 93 bp. The methylation status of DNA was determined in duplicate samples of three independent experiments.
Immunofluorescence labeling and confocal laser scanning microscopy (CLSM) of cells
Cover-slips with the captured cells were rinsed three times in phosphate buffer (PBS, pH 7.6) and fixed for 15 min in phosphate buffer supplemented with 3.3% (w/v) paraformaldehyde. Then cells were rinsed three times in PBS, pH 7.6, and permeabilized with 3.3% Triton X-100 for 15 min. The cells were blocked with phosphate buffer containing 5% (v/v) goat serum (DAKO) for 60 min at room temperature. Then, the cover-slips were rinsed and incubated with the indicated primary antibodies against H3K4Me3, H3K9Ac, H3K9Ac/S10Ph (Upstate Biotechnology Inc., Lake Placid, NY) and anti-cd34+-FITC (Miltenyi Biotec Inc., Bergisch Gladbach, Germany) for 90 min at 37°C and three times rinsed with PBS, pH 7.6. Finally, the cover-slips that needed were incubated with secondary antibodies, i.e. Alexa 564-coupled goat anti-rabbit or Alexa 488-coupled goat anti-rabbit Fab fragments (Molecular Probes, Eugene, OR) at a concentration 15 μg/ml for visualization.
For confocal imaging, we used a Bio-Rad Radiance 2100 and Radiance 2000MP (Carl Zeiss, Jena, Germany). Images were taken in sequence after inserting the signal enhancing lenses by activating channel 1 (blue); not used: Mai-Tai laser (815 nm), with dichroic beam -splitter 500DCLPXR, blocking filter BGG22 and emission filter D488/10; channel 2 (green): Argon laser (488 nm), no blocking filter and emission filter HQ545/40; and channel 3 (red): Argon laser (488 and 514 nm), no blocking filter and the emission filter E600 LP. The microscope was a Nikon Eclipse TE2000U (Tokyo, Japan), equipped with PlanApo DicH x60 oil immersion objective (NA 1.40).
For visualization of modified histones in CD34+, KG1 and NF three independent biological experiments were carried out. Through observation of the samples around 70-80% of the cells displayed a positive marking of modified histones. Images of representative 4–9 cells from each experiment for each histone modification were taken and summarized in the graphs of the Figures 4, 5, 6 and 7.
Data provided by fluorescence image analysis were not normally distributed, so Wilcoxon rank sum test was used as nonparametric alternative to the two-sample t-test used for independent samples. The Wilcoxon rank – sum test allows a hypothesis test of the equality of two samples medians. *P < 0.05 and **P < 0.01 were considered as statistically significant, and NS describes no significant change. The bar graphs in Figures 4, 5, 6 and 7 represent fold- enrichment of the modified histones relative to the control (CD34+ cells). Data is the mean ± SD from three independent experiments.
In this research, suit of custom image analysis functions were used. Functions have been implemented in Matlab™ environment (The MathWorks, Natick MA, USA) and were built based on our prototype for 2-Dimensional Electrophoresis gel image analysis .
The developed tools were used for fluorescent image analysis, i.e. image preprocessing, segmentation, fluorescent intensity data mining and statistical data analysis. During image preprocessing Gaussian image smoothing is performed for noise reduction. Purpose of segmentation is to acquire spot boundary that delineates cell area from background and other cells. Segmented cell area is used as region of interest (ROI) for spot volume calculations. During segmentation, all available cell layers, that were acquired from microscope, are used. Key tools in segmentation algorithm are symmetrical feature detector and Watershed transformation. Symmetrical feature detector generates map of second order symmetries by the use of the Johansson method . Watershed transformation is used for splitting of symmetry map. After isolation of individual cells, total fluorescent intensity of each cell was obtained.
The protein quantity V
in a cell is defined as the total fluorescent intensity in a segmented region of corresponding cell. The total intensity of an object is the sum of the intensities of all the pixels that make up the object:
After quantifying all cells, median values of total fluorescence intensities of each cell grouped by class were computed. The ratios between median values represent fold-change in protein expression. An increase of fluorescent intensity yields a positive fold-change and a decrease, accordingly a negative fold-change. Wilcoxon’s rank sum test was used to evaluate statistically significant changes.