Assessing Cellular Oxidative Stress and Viability

H₂DCFDA ROS assay was carried out according to established protocols, with some adjustment (Supplemental Methods) (6 (link)). Cell toxicity was measured via ATP depletion (CellTiter Glo, Promega) in THLE-2 cells (ATCC, CRL-2706), following 72 hours of exposure to the test compounds, and cell viability was assessed using XTT assay (Biological Industries) according to manufacturers protocols. CETSA was performed as previously described (53 (link)) with some modifications, using QR2 (Santa Cruz, sc-271665) and SOD1 (Santa Cruz, sc-17767) antibodies. SDS-PAGE and immunoblot imaging were carried out as previously described (55 (link)), using Tubulin (Sigma-Aldrich, SAB4500087), QR2 (Santa Cruz, sc-271665), NDUFA9 (AbCam, ab14713) and CD73 (Cell Signaling Technology, D7F9A) antibodies.

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Gould N.L., Scherer G.R., Carvalho S., Shurrush K., Kayyal H., Edry E., Elkobi A., David O., Foqara M., Thakar D., Pavesi T., Sharma V., Walker M., Maitland M., Dym O., Albeck S., Peleg Y., Germain N., Babaev I., Sharir H., Lalzar M., Shklyar B., Hazut N., Khamaisy M., Lévesque M., Lajoie G., Avoli M., Amitai G., Lefker B., Subramanyam C., Shilton B., Barr H, & Rosenblum K. (2023). Specific quinone reductase 2 inhibitors reduce metabolic burden and reverse Alzheimer’s disease phenotype in mice. The Journal of Clinical Investigation, 133(19), e162120.

Publication 2023

CellTiter Glo XTT assay NDUFA9

Antibodies Assay Biological Cd73 Cell viability Cells H2dcfda Immunoblot Promega Sds page Tubulin

Corresponding Organization :

Other organizations : University of Haifa, Weizmann Institute of Science, University of Windsor, Western University, McGill University, Montreal Neurological Institute and Hospital

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Variable analysis

independent variables

Test compounds

dependent variables

ROS (Reactive Oxygen Species) production
Cell toxicity
Cell viability
Protein thermal stability (CETSA)

control variables

THLE-2 cells (ATCC, CRL-2706)
Cell exposure time (72 hours)

controls

Positive control: not specified
Negative control: not specified

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