MCF7 and MDA-MB-231 breast cancer cell lines (ATCC; STR authenticated) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 100 Units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (PSG, Sigma-Aldrich) and 10% heat inactivated fetal bovine serum (FBS, First Link). MCF7/LMTK3 and MDA-MB-231/LMTK3, stably over-expressing LMTK3, were cultured in DMEM supplemented with 10% FCS, G418 (500 μg/ml; Invitrogen) and 1% PSG. All cells were incubated at 37 °C in humidified 5% CO2, and were frequently tested for mycoplasma contamination. Antibodies used: LMTK3 (sc-100418, Santa Cruz Biotechnology); LMTK3 (H00114783-M02, Abnova); β-actin (ab627, Abcam); phospho-histone H2A.X Ser139 (2577, Cell Signaling Technology); phospho-histone H2A.X Ser139 (9718, Cell Signaling Technology); ATM (sc-23921, Santa Cruz Biotechnology); phospho-ATM Ser1981 (4526, Cell Signaling Technology); KAP1 (ab10484, Abcam); phospho-KAP1 Ser824 (ab70369, Abcam). Doxorubicin hydrochloride (D1515) was purchased from Sigma-Aldrich.
Phospho histone h2a x ser139
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Histone H2A.X (Ser139) is a laboratory reagent that detects the phosphorylation of histone H2A.X at serine 139. This modification is a marker of DNA double-strand breaks.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (8 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Phospho p53 ser15, by Cell Signaling Technology (6 mentions)
Phospho chk1 ser345, by Cell Signaling Technology (6 mentions)
Phospho chk2 thr68, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (4 mentions)
Gapdh, by Merck Group (4 mentions)
Nocodazole, by Merck Group (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Phospho atm ser1981, by Cell Signaling Technology (3 mentions)
Phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (3 mentions)
Cleaved parp, by Cell Signaling Technology (3 mentions)
α tubulin, by Merck Group (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
647 far red, by Thermo Fisher Scientific (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Phospho atr ser428, by Cell Signaling Technology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 3 asp175, by Cell Signaling Technology (2 mentions)
Cyclin e1, by Cell Signaling Technology (2 mentions)
69 protocols using phospho histone h2a x ser139
1
Breast Cancer Cell Lines Protocol
2
Immunoblotting Analysis of Signaling Proteins
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Immunoblotting was performed essentially as described7 (link), except that the Westar ηC Ultra 2.0 chemiluminescence substrate (Cyanagen, Bologna, Italy) was employed. Rabbit monoclonal anti-ATP1B3 and rabbit polyclonal anti-transferrin receptor antibodies were purchased from Abcam (Cambridge, England). Mouse monoclonal anti β-catenin antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit monoclonal PI3 Kinase p100α, rabbit polyclonal AKT, rabbit polyclonal phospho-AKT (Ser473), rabbit monoclonal GSK3β, rabbit polyclonal phospho-GSK3β (Ser9) and phospho-Histone H2A.X (Ser139) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal anti-β-actin antibody was purchased from Sigma (St. Louis, MO, USA).
3
DNA Damage Detection via γH2AX Assay
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DNA damage was detected using Phospho-Histone H2A.X (Ser139) antibody for γH2AX (1:300-500, Cell Signaling Catalog Number 2577S). Nocodazole was purchased from Sigma-Aldrich. Secondary antibodies used were Alexa Fluor 488 (green), 594 (red) and 647 (far red) (1:1000, Life Technologies).
4
Evaluating Combination Treatments for Mia PaCa-2 Cells
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Mia PaCa-2 cells were seeded in an
8-well chamber slide with a density of 2,000 cells/well and incubated
for 48 h prior to treatment. Each well was then replaced with fresh
media containing 0.1% DMSO (v/v) and the following treatments: Pd0-resins (0.67 mg/mL);5p (300 nM); 1 (300 nM); or a combination of Pd0-resin + 5p (300 nM). The combination of 5p and Pd0 resins
were incubated for 24 h at 37 °C prior to treatment with cells.
Following 24 h of treatment, cells were fixed with paraformaldehyde
(4%, 20 min) and permeabilized cells (Triton X-100, 0.1%) subsequently
treated with rabbit monoclonal antibody against Phospho-Histone H2AX
(Ser139) (1:400, Cell Signaling Technologies, cat. no. 9718) for 1
h at room temperature. This was followed by 1 h of incubation at room
temperature with secondary Alexa Fluor 488 linked antibody (1:1000,
goat antirabbit, IgG, Life Technologies), Hoechst 33342 (1:8000, Life
Technologies), and Alexa Fluor 594 Phalloidin (1:500, Life Technologies).
Cells seeded in the chamber slide were imaged using an Olympus FV1000
microscope and merged using the ImageJ software (National Institutes
of Health).
8-well chamber slide with a density of 2,000 cells/well and incubated
for 48 h prior to treatment. Each well was then replaced with fresh
media containing 0.1% DMSO (v/v) and the following treatments: Pd0-resins (0.67 mg/mL);
were incubated for 24 h at 37 °C prior to treatment with cells.
Following 24 h of treatment, cells were fixed with paraformaldehyde
(4%, 20 min) and permeabilized cells (Triton X-100, 0.1%) subsequently
treated with rabbit monoclonal antibody against Phospho-Histone H2AX
(Ser139) (1:400, Cell Signaling Technologies, cat. no. 9718) for 1
h at room temperature. This was followed by 1 h of incubation at room
temperature with secondary Alexa Fluor 488 linked antibody (1:1000,
goat antirabbit, IgG, Life Technologies), Hoechst 33342 (1:8000, Life
Technologies), and Alexa Fluor 594 Phalloidin (1:500, Life Technologies).
Cells seeded in the chamber slide were imaged using an Olympus FV1000
microscope and merged using the ImageJ software (National Institutes
of Health).
5
Western Blot Analysis of Cell Signaling
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Cell lysates were prepared using RIPA buffer (9806, Cell Signaling) supplemented with protease and phosphatase inhibitors (11836153001 and 4906845001; Roche) according to manufacturer's instructions. Lysates were mixed with 4× Loading Buffer (928‐40004, Li‐Cor) and denatured at 95°C for 5 min prior to electrophoresis. SDS‐PAGE was performed with the NuPAGE® electrophoresis system (Thermo Fischer) including 4%–12% Bis‐Tris gels (Thermo Fischer, # NP0336BOX) and MES SDS Running Buffer (NP0002; Thermo Fisher) according to the manufacturer's instructions. Western blotting onto nitrocellulose membranes (10600002; GE Healthcare) was performed inside an XCell II™ Blot Module (Thermo Fischer) for 1 h at 30 V constant (25 mM Tris‐BASE; 192 mM Glycine, 20% (v/v) Methanol transfer buffer). Membranes were washed with TBS (15 mM Tris–HCl pH 7.6; 136 mM NaCl) and blocked with TBS Blocking Buffer (927‐50000, Li‐Cor). Primary antibodies were incubated at 4°C overnight according to manufacturer's instructions. The following antibodies were used: Phospho‐Histone H2A.X (Ser139) (2577, Cell Signaling), Cyclin D1 (2978, Cell Signaling), Lamin B1 (13435, Cell Signaling), p16Ink4a (80772, Cell Signaling), p21Cip1 (2948, Cell Signaling), GAPDH (2118, Cell Signaling), FLAG (740001, Thermo Fischer), pMLC (3675, Cell Signaling), and alpha‐SMA (ab5694, Abcam).
6
Damulin B Modulates Apoptosis Signaling
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Dulbecco’s modified Eagle’s medium (DMEM) powder was purchased from Gibco (Grand Island, NY, USA). Antibodies against Bax, Bad, cleaved PARP-1, Bcl-2, β-actin, and HA-Tag, and small interfering RNA (siRNA) against AMPKα1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against AMPKα1, cleaved caspase-7, cleaved caspase-3, phospho-ATR (Ser428), and phospho-histone H2A.X (Ser139) were purchased from Cell Signaling Technology (Danvers, MA, USA). The DNA transfection reagent and siRNA transfection reagents were purchased from Thermo Fisher (Waltham, MA, USA). Damulin B was obtained from ChemFaces Biochemical (Wuhan, China).
7
Immunohistochemical Analysis of Lung Tissue
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Lungs were perfused with 10% formalin, stored in fixative overnight, and embedded in paraffin. For further staining (hematoxylin/eosin and immunohistochemisty), sections of 5 μm were cut. Primary antibodies from Cell Signaling Technologies (Danvers, MA, USA) were used at the listed dilutions: phospho-Histone H2A.X (Ser139) (#20E3) (1:400), Cleaved Caspase-3 (Asp175) (#5A1E) (1:800). The Ki67 antibody (16A8) was purchased from BioLegend (San Diego, CA, USA) and used at a dilution of 1:50. Positive cells were identified visually in each high-power field (40× magnification). A total of 3–5 fields were observed per tumour.
8
Western Blot Analysis of DNA Damage Response
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The details are described in previous report [32 (link)]. For Western blotting, the used antibodies were as following: p21 (OP79; Oncogene Science), p-ATM (Ser1981) (sc-47739; Santa Cruz, CA, USA), p53 (sc-98, Santa Cruz), cleaved caspase-3 (#9661; Cell Signaling, Danvers, MA, USA), phospho-Chk2 (Thr68) (#2197; Cell Signaling), Chk2 (#05-649; EMD Millipore, Temecula, CA, USA). LC3A/B (#12741; Cell Signaling), Chk1 (#2345; Cell Signaling), phospho-Chk1 (Ser345) (#2348; Cell Signaling), phospho-Histone H2A.X (Ser139) (#9718; Cell Signaling), ATM (GTX70103; GeneTex, Irvine, CA, USA), GAPDH (#2118, Cell Signaling), PARP (#9542; Cell Signaling), phospho-p53 (Ser15) (#9284; Cell Signaling). phospho-ATR (Ser428) (#2853; Cell Signaling). PAI-1 (#612024; BD Transduction Laboratories, Lexington, KY, USA).
9
Antibody Sources for Cell Cycle Regulation
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Antibodies were purchased from the following sources: Cdt1 (Cat# 8064, at least two different lots were used in this study), Chk1 (Cat# 2345), phospho-Chk1 S345 (Cat# 2341), Cyclin E1 (Cat#4129), MAPKAPK-2 (Cat#), Phospho-MAPKAPK-2 T334 (Cat#3007), phospho-Histone H2A.X Ser139 (Cat#9718) from Cell Signaling Technologies; hemagglutinin (HA) (Cat#11867423001) from Roche; Geminin (Cat#sc-13015), Cdc6 (Cat#sc-9964), MCM6 (Cat#sc-9843), Cyclin A (Cat#sc-596), Cyclin B1 (Cat#sc-245) and CDK2 (Cat#sc-163) from Santa Cruz Biotechnology; MCM4 (Cat#3728) from Abcam. MCM2 antibody (Cat#A300-191A) used for co-immunoprecipitation experiment was purchased from Bethyl Laboratories. MCM2 antibody (BD Biosciences, Cat#610700) was used for analytical flow cytometry. Serum to detect CDK1 was a gift from Y. Xiong (University of North Carolina), and MPM2 antibody was a gift from R. Duronio [82 (link)] (University of North Carolina). The phosphospecific Cdt1 antibody was described in Chandrasekaran et al [17 (link)].; the third and fourth test bleeds are active for Cdt1 immunoprecipitation. Alexa 647-azide and Alexa-488-azide used in flow cytometry analyses was purchased from Life Technologies, and secondary antibodies for immunoblotting and immunofluorescence were purchased from Jackson ImmunoResearch.
10
Comprehensive Antibody Immunodetection Protocol
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Antibodies recognizing the Flag tag (Sigma,F1804), SETX (Novus, NBP1-94712), RNA polymerase I (RPA194 (C-1) (Santa Cruz, sc-48385), fibrillarin (G-8) (Santa Cruz, sc-374022), nucleophosmin (NPM1, Forties Life Sciences A302-403A-T), C1QBP/P32 (Proteintech, 24474-1-AP), CHCHD2 (Proteintech, 66302-1-lg), GAPDH (Sigma-Aldrich, G9545), U2AF65 (Sigma-Aldrich, U4758), H3 (Abcam, ab8898), phospho-histone H2A.X(Ser139) (Cell signaling, 2577), and MitoTracker Red CMXRos (Invitrogen, M7512) were obtained from the indicated vendors. Human RIEP rabbit polyclonal antibodies were generated at Bethyl Laboratories/Fortis Life Sciences. RIEP-1 and RIEP-216 antibodies recognize peptides derived from the RIEP N terminus or C terminus, respectively. IGPAL-CA-630 (Sigma, I8896), cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Roche, 04693159001), and Pierce™ Protein A/G Magnetic Beads (Thermo Fisher scientific, 88802) were purchased from indicated vendors.
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