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5 protocols using phosphorylated histone h3

1

Comprehensive Characterization of HeLa Cell Responses

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HeLa cervical cancer cells were purchased from Highveld Biological, Johannesburg, South Africa. RPMI 1640 cell culture medium and fetal bovine serum was purchased from GE Healthcare Life Sciences (Logan, UT, USA). Trypsin-EDTA, Dulbecco’s phosphate buffered saline (DPBS) with Ca2+ and Mg2+ and Dulbecco’s phosphate buffered saline (DPBS) without Ca2+ and Mg2+ were purchased from Lonza (Wakersville, MA, USA). Trypan blue, bisBenzamide H 33,342 trihydrochloride (Hoechst 33342), cisplatin, penicillin/streptomycin and bovine serum albumin fraction V were purchased from Sigma-Aldrich (St. Louis, MI, USA). NucRedTM Live 647, CellRox® Orange reagent, Lysotracker™ Deep Red and Tetramethylrhodamine ethyl ester (TMRE) were purchased from Molecular Probes®-Life Technologies-Thermo Fisher Scientific (Logan, UT, USA). Annexin V-FITC/PI kit was purchased from MACS Miltenyi Biotec (Cologne, Germany). Cleaved caspase 3 (Asp175) (D3E9) Rabbit mAb, Cleaved caspase 8 (Asp391) (18C8) Rabbit mAb, Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor® 647 conjugate), Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor® 488 conjugate) and Phosphorylated Histone H3 were purchased from Cell Signaling Technology (Danvers, MA, USA).
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2

Immunohistochemistry for Cell Markers

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We performed immunohistochemical stainings following standard protocols using antibodies against Ki67 (15580, Abcam), phosphorylated-histone H3 (9701, Cell Signaling Technology) and Cleaved Caspase 3 (9664, Cell Signaling Technology) were used.
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3

Microtubule Dynamics in PANC-1 Cells

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70,000 PANC-1 cells were plated on a 6 well plate with a coverslip placed on the bottom, allowed to adhere overnight and then treated with either 10, 100 or 1000 nM leiodermatolide, 100 nM vinblastine, 100 nM nocodazole or their respective vehicle controls for 24 hours. Cells were labeled with antibodies against α tubulin (Sigma Aldrich, St. Louis, MO), phosphorylated Histone H3 (Cell Signaling Technology, Danvers, MA) or counterstained with propidium iodide to label the nuclei. After staining, the cover slip was dried and placed on a slide with 1 drop slowfade (Cat #536936, Invitrogen, Carlsbad, CA) and sealed with clear nail polish. Images were captured using an Olympus confocal microscope FVX (FV-200) system equipped with the Fluoview software.
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4

Immunohistochemistry for Cell Markers

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We performed immunohistochemical stainings following standard protocols using antibodies against Ki67 (15580, Abcam), phosphorylated-histone H3 (9701, Cell Signaling Technology) and Cleaved Caspase 3 (9664, Cell Signaling Technology) were used.
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5

Protein Profiling by Immunoblotting

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Protein extraction, two-dimensional polyacrylamide gel electrophoresis, and transfer to nitrocellulose membranes were performed as previously described.10 (link),11 (link) The antibody against CADM1 was purchased from Medical & Biological Laboratories (Woburn, MA). The antibody against SPC25 was purchased from Proteintech (Rosemont, IL). The antibody against CD133 was purchased from Abcam (Cambridge, United Kingdom). The antibodies against E-cadherin, VIM, AKT, phosphorylated-AKT, ERK, phosphorylated-ERK, phosphorylated-histone-H3, and cleaved PARP were purchased from Cell Signaling Technology (Danvers, MA). The antibody against glyceraldehyde-3-phosphate dehydrogenase was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
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