Journal: Scientific Reports
Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53
Figure Lengend Snippet: Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P
Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.
Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Staining, Double Staining, FACS, Real-time Polymerase Chain Reaction, Transfection, Cell Viability Assay, MTT Assay