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G; Millipore Temecula, CA, USA 07-030); anti-H3K9ac (IF dilution 1:200, ChIP 2.4 g; Abcam, Cambridge, MA, USA ab10812); anti-H3K9me2 (IF dilution 1:200, ChIP 3 g; Abcam, ab1220), anti-H3K9me3 (WB dilution 1:250; Abcam, ab8898; IF dilution 1:200, ChIP 3 g; Diagenode, Denville, NJ, USA CS-056-050); anti-CENP-A (IF dilution 1:200, ChIP 5 g; Abcam, ab13939); anti-HP1 (IF dilution 1:100, ChIP 4 g; Abcam, ab77256); anti-HP1 (IF dilution 1:100, ChIP 4 g; Abcam, ab10811); anti-GFP (ChIP 4 g; Abcam, ab290); and anti-H3 N-terminal (ChIP 1.5 g; Sigma, St. Louis, MO, USA, H9289-200 l).Cell viability (IC50) after TSA treatmentPBS (pH 7.4) for 10 min, followed by permeabilization in 0.4 IGEPAL (Sigma CA-630) in PBS for 10 min at room order AZD3759 temperature and incubated with 0.5 BSA blocking buffer. For each pair of primary antibodies, the optimal order of addition was determined in preliminary experiments. With the exception of ACA, which was visualized with a fluorescein-conjugated secondary antibody, the fluorophores on the secondary antibodies were Alexa Fluor 488-conjugated (Invitrogen, Life Technologies, M ico; A11001 anti-mouse, A11008 anti-rabbit, and A11078 anti-goat) for green fluorescence and Cy3-conjugated (Millipore, Temecula, CA, USA; AP124C anti-mouse and AP1132C anti-rabbit) for red fluorescence. Following incubation with the primary and secondary antibodies, DNA was stained with DAPI. The cells were observed by fluorescence microscopy using a Zeiss Axio Imager A2 (Carl Zeiss? Germany); the images were analyzed using the software AxioVision 4.8 (Carl Zeiss? Germany). The cells were also observed by laser confocal microscopy using a Zeiss LSM 710 Duo (Carl Zeiss? Germany); the images were analyzed using the Zen 2008 software (Carl Zeiss? Germany).TSA treatmentHuman WI-38 and HCT116 cells were obtained from ATCC (CCL-75 and CCL-247). All cell lines were tested and authenticated and were maintained in Eagle’s Minimum Essential Medium (EMEM; ATCC) and McCoy (Gibco) medium, respectively, supplemented with 10 fetal bovine serum (Gibco) and antibiotics; the cells were incubated at 37 in a 5 CO2 atmosphere. The cells were treated with TSA (Sigma, T8552-5MG) at 37 for 24 and 48 h. IC50 concentrations were determined by plating 80,000 cells in 24-well dishes containing 0.5 ml of medium and incubating overnight at 37 ; TSA was added when cultures reached 80 confluence. Cells were washed with PBS and fixed with 70 ethanol at -20 , then washed in PBS and stained with 1 crystal violet. After washing, the stain was solubilized in 33 acetic acid, and the absorbance was determined in an ELISA reader at 570 nm. The analyses were performed in triplicate in three independent experiments. The IC50 values were calculated by linear regression analysis of the dose-response data using the points in the exponential region of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28298493 the curve. The concentrations used for the TSA experiments were below the IC50: 4.9 M for HCT116 cells and 9.4 M for WI-38 cells.ImmunofluorescenceExponentially growing HCT116 and WI-38 cells were cultured on glass coverslips for 24 and 48 h in medium containing 1 M/ml TSA (Sigma, T8552-5MG), with daily media changes. The cells were washed with PBS, fixed with PFA, and used for immunofluorescence analysis, as described above. For treated chromatin isolation, WI-38 and HCT116 cells were cultured on 100-mm culture plates and treated in the same manner as the cell cultures grown on glass coverslips. We used 1 M TSA for 24 h and 48 h because a.