A set of four human Fas-specific siRNAs were obtained from Qiagen. W620-5FU-R cells were transiently transfected with these set of four siRNAs and analyzed for Fas expression by RT-PCR. One Fas siRNA (Cat # S100000637) was used to silence Fas expression for functional studies. SW620-5FU-R cells were seeded onto 24-well plates at 2.5×105 cells/well with scramble (200 nM) or Fas (200 nM) siRNA for 24 hours. Cells were then harvested and replated at 2×104 cells/well. Cells were then treated with or without verticillin A (20 nM) and MegaFasL (250ng/mL) for 24 hours. Cells were collected and analyzed for cell death by staining with Annexin V and PI. For sensitivity to 5-FU and verticillin A, cells were treated with or without verticillin A (20nM) and 5-FU (1 μg/mL) for 72 hours and analyzed for cell death by staining with Annexin V and PI and flow cytometry analysis. G9a-, SUV39H1- and SUV39H2-specific siRNAs were obtained from Santa Cruz Biotech (Cat# sc-43777, sc-38463 and sc-106822). SW620-5FU-R cells were transfected as described above. Cells were then analyzed for G9a, SUV39H1, SUV39H2 and Fas expression using real-time RT-PCR. Acidic extracts were also prepared from these cells and analyzed by Western blotting using anti-H3K9me3 antibody (Abcam).
Anti h3k9me3 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-H3K9me3 antibody is a lab equipment product designed to detect the trimethylation of lysine 9 on histone H3. This epigenetic modification is associated with transcriptional repression and heterochromatin formation.
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Lab products found in correlation
Anti rabbit igg hrp, by Bio-Rad (2 mentions)
Anti histone h3, by Abcam (2 mentions)
Anti flag m2 affinity gel resin, by Merck Group (2 mentions)
Flag peptide, by Merck Group (2 mentions)
90i microscope, by Zeiss (2 mentions)
Prolong with dapi, by Thermo Fisher Scientific (2 mentions)
Lsm170, by Zeiss (2 mentions)
4 well chamber slide, by Merck Group (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Chromatin immunoprecipitation chip assay kit, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Abi prism 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Rna polymerase 2, by Santa Cruz Biotechnology (1 mentions)
H3k9ac, by Cell Signaling Technology (1 mentions)
Fitc labeled goat anti rabbit igg, by Beyotime (1 mentions)
Ds ri1 digital camera, by Nikon (1 mentions)
Alexa fluor cy3 labeled goat anti mouse igg, by Beyotime (1 mentions)
Eclipse ti s microscope, by Nikon (1 mentions)
20 protocols using anti h3k9me3 antibody
1
Silencing Fas Expression Enhances Chemosensitivity
2
Chp1CD Nucleosome-Binding Assays
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Chp1CD wild-type and mutant nucleosome-binding assays were performed as described earlier for the Chp1CD—H3KC9me3 MLA Nucleosome complex formation in 20 mM HEPES pH 7.5, 100 mM KCl, 0.5 mM DTT, 40 mM imidazole. Resins and INPUTs were run on SDS-polyacrylamide gel electrophoresis 15% acrylamide gels. All samples were then analyzed by immunoblot with anti-H3 histone (AbCam, Cambridge, UK, 1:1000), or anti-H3K9me3 Antibody (AbCam, 1:1000), anti-goat IgG-HRP (BioRad, 1:3000) anti-rabbit IgG-HRP (BioRad, Munich, Germany, 1:3000).
3
Epigenetic Analysis of Reproductive Tissues
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For methylation analysis of oocytes, sperm, and placentas, 160 oocytes were directly subjected to proteinase K digestion and bisulfite treated, DNA of sperm was isolated from paternal mature sperms and (C57BL/6 × DBA and DBA × C57BL/6), and placental DNA was isolated from E15.5 F1-generation placenta. Then the DNA (sperm, placenta and chorionic plate) and treated oocyte samples were analyzed using an EZ DNA methylation-Gold kit (Zymo Research, USA), according to the manufacturer’s instructions. Chromatin immunoprecipitation assays were performed using a ChIP assay kit (Upstate Biotechnology, USA) following the manufacturer’s instructions. The bisulfite-treated DNA was amplified by nested PCR using 2× GoldStar Best MasterMix (Cowin Biotech, China). The PCR products were sequenced using an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems, USA). Nested PCR primer sequences were: forword, 5′-TAG GTT TTT GGG AGA GGA TTG T-3′; reverse-out, 5′-ATA ATT AAT TTA GGG GTG GAG AAG G-3′; reverse-in, 5′-AAT TCC CAA CTA TCA AAA ACA AAA C-3′. The antibody were used as following, anti-H3K4me3 and H3K4me1 (Cell Signaling Technology, USA), anti-H3K9me3 antibody (ab8898, Abcam, United Kingdom), normal rabbit IgG (Santa Cruz Biotechnology, USA).
4
Chromatin Immunoprecipitation for Histone Modifications
5
Immunofluorescence Analysis of H3K9me3 and Cell Counts in Blastocysts
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Zona pellucida-free embryos were washed 3 times in PBS, fixed in freshly prepared 4% paraformaldehyde in PBS, permeabilized in 1% Triton X-100 in PBS, and incubated in blocking solution (1% bovine serum albumin in PBS) for 1 h. For immunolabeling, the embryos were incubated overnight at 4 °C with an anti-H3K9me3 antibody (1:800 dilution; ab8898, Abcam) followed by 3 washes with PBS and a 1-h incubation with a FITC-labeled goat anti-rabbit IgG (1:1000 dilution; Beyotime, Shanghai, China) secondary antibody. The samples were then washed and counterstained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime) to label DNA. Fluorescence was analyzed using a Nikon Eclipse Ti-S microscope equipped with a 198 Nikon DS-Ri1 digital camera (Nikon, Tokyo, Japan).
The cell numbers in blastocysts were estimated by counting nuclei stained using DAPI, and the number of TE cells was determined by counting nuclei positive for CDX2. The cell number of ICM was estimated as the total number of nuclei minus the number of TE nuclei (41 (link), 42 (link)). The blastocyst was incubated with anti-CDX2 (1:200, BioGenex Inc., San Ramon, CA) for detecting the TE, and the secondary antibody was Alexa Fluor Cy3-labeled goat anti-mouse IgG (1:1000 dilution; Beyotime, Shanghai, China).
The cell numbers in blastocysts were estimated by counting nuclei stained using DAPI, and the number of TE cells was determined by counting nuclei positive for CDX2. The cell number of ICM was estimated as the total number of nuclei minus the number of TE nuclei (41 (link), 42 (link)). The blastocyst was incubated with anti-CDX2 (1:200, BioGenex Inc., San Ramon, CA) for detecting the TE, and the secondary antibody was Alexa Fluor Cy3-labeled goat anti-mouse IgG (1:1000 dilution; Beyotime, Shanghai, China).
6
Histone H3K9me3 ChIP-Seq in 5-FU-Resistant Cells
7
Western Blotting for Protein Analysis
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Harvested cells were lysed using Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA). Protein concentrations were quantified using the BCA Protein Assay (Pierce) according to manufacturers instructions. Around 20–40 µg of protein mixed with a 6x loading dye containing β-mercaptoethanol was boiled for 10 min before it was run on a 4–15% gradient gel. Protein was transferred to a nitrocellulose membrane using the Trans-Blot Turbo (Bio Rad, Hercules, CA, USA) and membranes blocked for 30 min – 1 h in 3% BSA. Membranes were probed overnight with anti-FLAG M2 at 1:2000 dilution (Sigma-Aldrich). Anti-ACTIN antibody at 1:10 000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a loading control.
To assess global H3K9 trimethylation by Western blotting, nuclear proteins were collected from cells transfected with KLLN plasmid or siRNA using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce). Protein concentration was measured using the BCA Protein assay and 15–25 µg of protein was used for immunoblotting, done as described above. Membranes were probed overnight with anti-H3K9me3 antibody (1:1000) (Abcam Inc., Cambridge, MA, USA, cat# ab8898). Anti-PARP-1 antibody (1:1000) (Santa Cruz Biotechnology, cat# sc-8007) was used as a nuclear loading control.
To assess global H3K9 trimethylation by Western blotting, nuclear proteins were collected from cells transfected with KLLN plasmid or siRNA using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce). Protein concentration was measured using the BCA Protein assay and 15–25 µg of protein was used for immunoblotting, done as described above. Membranes were probed overnight with anti-H3K9me3 antibody (1:1000) (Abcam Inc., Cambridge, MA, USA, cat# ab8898). Anti-PARP-1 antibody (1:1000) (Santa Cruz Biotechnology, cat# sc-8007) was used as a nuclear loading control.
8
Clr4 Binding Assay on Nucleosomes
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The binding assay of all Clr4 constructs and the three types of nucleosomes were conducted as the following: 0.5 μg of protein were bound to 15 μl of anti-FLAG M2 affinity gel resin (Sigma-Aldrich) in binding buffer (50 mM phosphate buffer pH 6.8, 150 mM NaCl, 1 mM DTT) for 20 min at 4°C. The resin was washed once with the binding buffer and 2 mg of nucleosomes were then added to reach a final volume of 40 μl. After 1 h incubation on ice, the resin was centrifuged and the flow-through was collected. The flow-through, resins and inputs were analyzed on SDS-PAGE 15% acrylamide gels then silver staining (0.1% AgNO3). All samples were further analyzed with western blot using anti-H3 histone (AbCam, 1:1000), anti-H3K9me3 Antibody (AbCam, 1:1000), anti-goat IgG-HRP (BioRad, 1:3000) and anti-rabbit IgG-HRP (BioRad, 1:3000).
The Clr4-H3KC9me3 nucleosome complexes (Clr4 FL, Clr4 CD and Clr4 1–191 constructs) were prepared as mentioned above using 5 μg protein and 10 μg nucleosomes and eluted after incubation with FLAG peptide (Sigma-Aldrich).
The Clr4-H3KC9me3 nucleosome complexes (Clr4 FL, Clr4 CD and Clr4 1–191 constructs) were prepared as mentioned above using 5 μg protein and 10 μg nucleosomes and eluted after incubation with FLAG peptide (Sigma-Aldrich).
9
Immunofluorescence Staining of H3K9Me3
10
Chromatin Immunoprecipitation of H3K9me3
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