Cisplatin-Induced DNA Damage Response Signaling

Control and IPF fibroblasts (n=8, each) used for cell viability assay (6 × 10⁵ cells/60 mm cell culture dish) were treated with cisplatin for various time points. Cells were lysed with 1x cell lysis buffer (Cell Signaling technology, Beverly, MA) containing protease inhibitor (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor (Research Products International Corp., Mount Prospect, IL), and western blotting was performed as previously described (15 ,16 (link),17 (link)). The antibodies used for our study are: γH2AX (PRID: AB_2118009, Cell Signaling Technology) (21 (link)), H2AX (RRID: AB_10694556, Cell Signaling technology) (22 (link)), RAD51 (RRID: AB_2042762, Abcam, Cambridge, UK) (11 (link)), BRCA2 (RRID: AB_2259370; R&D systems, Minneapolis, MN) (11 (link)), pXRCC1 (phosphorylated at S518, T519, and T523; catalog No. ab195205; Abcam) (21 (link)), XRCC1 (RRID:AB_10985840; ThermoFisher Scientific, Pittsburgh, PA) (23 (link)), CK2α (catalog No. 2656; Cell Signaling technology)(24 (link)), PUMA (RRID:AB_10987708; Santa Cruz Biotechnology, Santa Cruz, CA)(25 (link)), or GAPDH (RRID:AB_627679; Santa Cruz Biotechnology) (26 (link)). The protein bands on a membrane were then detected by ECL solution (ThermoFisher Scientific) using Chemi Doc-IT2 image analyzer (UVP BioImage systems, Upland, CA), and quantified using VisionWorks LS program (UVP BioImage systems).

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Im J, & Nho R.S. (2019). Fibroblasts from patients with Idiopathic Pulmonary Fibrosis are resistant to cisplatin-induced cell death via enhanced CK2-dependent XRCC1 activity. Apoptosis : an international journal on programmed cell death, 24(5-6), 499-510.

Publication 2019

1x cell lysis buffer protease inhibitor γH2AX RAD51 BRCA2 XRCC1 GAPDH ECL solution

Antibodies Assay Brca2 Buffer Cell Cell viability Cells culture Cisplatin Ck2α Dish Fibroblasts Gapdh Membrane protein Phosphatase Protease inhibitor Puma Xrcc1

Corresponding Organization :

Other organizations : University of Minnesota

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Variable analysis

independent variables

Treatment with cisplatin for various time points

dependent variables

Cell viability

control variables

Cell type (Control and IPF fibroblasts)
Number of cells (6 × 10^5 cells/60 mm cell culture dish)
Cell lysis buffer and protease/phosphatase inhibitors
Western blotting procedure
Antibodies used (γH2AX, H2AX, RAD51, BRCA2, pXRCC1, XRCC1, CK2α, PUMA, GAPDH)

controls

No positive or negative controls were explicitly mentioned in the protocol.

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