P h2ax

Manufactured by Abcam

Sourced in United Kingdom

P-H2AX is a laboratory equipment product that detects and measures the presence of phosphorylated histone H2AX, a marker of DNA double-strand breaks. This product provides a quantitative assessment of DNA damage in cells.

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6 protocols using p h2ax

Western Blot Analysis of Apoptosis Markers

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Antibodies to CHOP (1∶500, CST: Cell Signaling Technology, Danvers, MA), DR5 (1∶500; CST), cleaved caspase-3 (1∶250; CST), caspase-8 (total form), cleaved caspase-8 (1∶250; CST), XIAP (1∶500, CST), Survivin (1∶500; CST), Bcl2 (1∶500; CST), c-FLIP (1∶500; CST), p-p53 (serine 15) (1∶500, CST), p-CHk1 (Serine 345) (1∶500; CST), p-H2AX (Serine 15), DR4 (1∶500, Abcam) Actin (1∶2000, Sigma Aldrich), BAX (1∶500; CST), PARP-1 (1∶1000; CST) and cleaved PARP (Asp214, 1∶1000; CST) were used. Western Blotting was performed as described previously [11] (link).

Karpel-Massler G., Pareja F., Aimé P., Shu C., Chau L., Westhoff M.A., Halatsch M.E., Crary J.F., Canoll P, & Siegelin M.D. (2014). PARP Inhibition Restores Extrinsic Apoptotic Sensitivity in Glioblastoma. PLoS ONE, 9(12), e114583.

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Comprehensive Protein Expression Analysis

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Cells were collected and lysed 12 h after transfection. Proteins were detected by a standard protocol using the following primary antibodies: cleaved PARP (Cell Signaling, cat#: 5625), cleaved caspase-3 (Abcam, cat#: ab32042), pH2AX (S139; Abcam, cat#: ab81299), pChk1 (S345; Abcam, cat#: ab58567), Chk1 (Abcam, cat#: ab40866), pChk2 (T68; BOSTER, cat#: P00277), Chk2 (Abcam, cat#: ab109413), p53(Abcam, cat#: ab26), pH2AX (Y142; Bio-Rad, cat#: AHP3045), pHER2 (Y11221/1222; Abcam, cat#: ab131102), HER2 (Abcam, cat#: ab134182), and β-Actin (Abcam, cat#: ab115777). Horseradish peroxidase (HRP)-labeled sheep anti-rabbit secondary antibody (Jackson, cat#:111–035-003) was incubated at room temperature for 2 h. The film was placed in a fully automated chemiluminescence instrument (Tanon 5200) for imaging, and the optimal image was saved for analysis (analysis system: TanonImage).

Yang X., Xu Y., Fu J, & Shen Z. (2023). Nanoparticle delivery of TFOs is a novel targeted therapy for HER2 amplified breast cancer. BMC Cancer, 23, 680.

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Western Blot Analysis of Signaling Proteins

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Cells were lysed in either a sodium dodecyl sulfate (SDS) lysis buffer: a 1:3 mixture of buffer I (5% SDS, 0.15 M Tris-HCl [pH 6.8], 30% glycerol) and buffer II (25 mM Tris-HCl [pH 8.3], 50 mM NaCl, 0.5% NP-40, 0.1% SDS, 1 mM EDTA) or Urea 8 M, containing 0.5 mM N-ethylmaleimide (NEM), 0.5 mM NaF and 2 mM sodium Na₃VO₄ and protease inhibitors (Sigma). After lysis, an equal amount of protein for each sample was resuspended in denaturing sample loading buffer, separated on SDS-PAGE gel and immunoblotted with the indicated antibodies. The following antibodies were used: SMAD4, CHK1, p-CHK1 and βTRCP1 (Cell Signaling Technology), p63, pH2Ax, Flag and GAPDH (Abcam), HPV16 E7, p53, H3 and Rad51 (Santa Cruz Biotechnology), and Vinculin (Merck Millipore). Membranes were then incubated with the appropriate horseradish peroxidase (HRP) secondary antibodies and the signal was acquired with Chemidoc (Bio-Rad). Densitometric analysis of the intensity of the protein bands were preformed using ImageJ software (Rasband W.S., ImageJ, U. S. National Institutes of Health), and standardized to the housekeeper levels.

Citro S., Miccolo C., Medda A., Ghiani L., Tagliabue M., Ansarin M, & Chiocca S. (2022). HPV-mediated regulation of SMAD4 modulates the DNA damage response in head and neck cancer. Journal of Experimental & Clinical Cancer Research : CR, 41, 59.

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Molecular Mechanisms of DOX-Induced DNA Damage

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DOX was provided by Beijing Huafeng United Technology (Beijing, China). Antibodies against GAPDH, ATM, p-ATM, H2AX, p-H2AX, YES1, p-YES1, p53, p-p53, AMPKα, p-AMPKα were purchased from Abcam (Cambridge, UK). Dulbecco’s modified Eagle’s high glucose medium and Dulbecco’s modified Eagle’s medium were provided by GIBCO (Grand Island, New York, NY, USA). Dual-Luciferase Reporter Assay System Kit (Thermo Fisher Scientific, Waltham, MA, USA) and pSI-CHECK2 vector were provided by Hanbio Biotechnology (Wuhan, China). EntiLink™ 1st Strand cDNA Synthesis Kit and EnTurbo™ SYBR Green PCR SuperMix Kit were purchased from ELK Biotechnology (Wuhan, China).

Wang L., Pan T., Wang Y., Yu J., Qu P., Chen Y., Xin H., Wang S., Liu J, & Wu Y. (2022). Effect of Nanoparticles of DOX and miR-125b on DNA Damage Repair in Glioma U251 Cells and Underlying Mechanisms. Molecules, 27(19), 6201.

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Molecular Mechanisms of Necroptosis

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Shikonin, Cyclosporine A (CsA), Nec-1, GSK872 and MnTBAP were purchased from selleckchem company (Houston, TX). Shikonin was dissolved in PBS to a storage concentration of 50 mmol/L. The primary antibodies against the following proteins RIP1, p-RIP1, RIP3, p-RIP3, Cyclophilin A, AIF, DKAPKcs, p-DNAPKcs, ATM, p-ATM, p-H2AX, H2A, NRF2, p-NRF2 antibodies were all purchased from Abcam company (Cambridge, MA). Anti-β-Actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Other reagents were purchased from Sigma (St. Louis, MO).

Wang X., Fan L., Wang X., Luo T, & Liu L. (2022). Cyclophilin A contributes to shikonin-induced glioma cell necroptosis and promotion of chromatinolysis. Scientific Reports, 12, 14675.

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Tumor Regression and Toxicity Assessment

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Tumor regression was graded according to decreased cancer cellularity and increased fibrous tissue (grade: 0, no regression; 1, minimal; 2, mild; 3, moderate; 4, marked; 5, severe) on hematoxylin–eosin-stained tissue sections. Intestinal toxicity (apoptotic cells in intestinal crypts of the jejunum) and bone marrow toxicity (decreased cellularity) were graded according to a standard toxicologic pathology scale (grade: 0, no change; 1, minimal; 2, mild; 3, moderate; 4, marked; 5, severe).
DNA damage was assessed through immunohistochemical staining for p53 binding protein 1 (53BP1 [Novus Biologicals]), phospho-histone gamma-H2AX protein (pH2AX [Abcam]) and RAD51 (Abcam) (further details in Supplementary methods). Quantification of the staining in tumors was performed using an H-score method (proportion of positive-labelled cells × staining intensity [grade: 0, no staining; 1, minimal; 2, moderate; 3, strong]). The H-score scale ranged from 0 (no staining) to 300 (strongest intensity of labeling in all cells [100%]). pH2AX immunostaining was also graded 0–5 to assess toxicity in the jejunum.

Barbier S., Beaufils B., de Miguel R., Reyre M., Le Meitour Y., Lortie A., de Boisferon M.H., Chaumeron S., Espirito A., Fossati L., Lagarde P., Klinz S., Thiagalingam A., Lezmi S, & Meyer-Losic F. (2023). Liposomal Irinotecan Shows a Larger Therapeutic Index than Non-liposomal Irinotecan in Patient-Derived Xenograft Models of Pancreatic Cancer. Oncology and Therapy, 11(1), 111-128.

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