Sirt1 Inhibitor Screening in MEFs

Sirt1 WT and KO MEFS cells were treated with 10 μM EX-527, 12 μM S1th 13 (6), 20 μM S1th 12 (5), 30 μM S1th 10 (7) and 30 μM SirReal2 [Sirt2 inhibitor, synthesized according to (62 (link))] for 48 h. Oxidative stress was induced using 10 μM Camptothecin 2 h. Whole-cell extracts were performed according to the Dignam protocol (63 (link)). Primary antibodies used for the western blot were anti-H2AX and anti-γH2AX (ab11175 and ab2893 resp., Abcam, UK). Densiometric analysis of the western blots was performed with Quantity One software (Bio-Rad Laboratories, Inc., USA).

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Wössner N., Alhalabi Z., González J., Swyter S., Gan J., Schmidtkunz K., Zhang L., Vaquero A., Ovaa H., Einsle O., Sippl W, & Jung M. (2020). Sirtuin 1 Inhibiting Thiocyanates (S1th)—A New Class of Isotype Selective Inhibitors of NAD+ Dependent Lysine Deacetylases. Frontiers in Oncology, 10, 657.

Publication 2020

ab11175 ab2893 Quantity One software

Antibodies Camptothecin Cell extracts Cells Oxidative stress Resp Sirreal2 Sirt1 Sirt2 Western blot Western blots analysis

Corresponding Organization : University of Freiburg

Other organizations : Martin Luther University Halle-Wittenberg, Josep Carreras Leukaemia Research Institute, Leiden University Medical Center

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Variable analysis

independent variables

10 μM EX-527
12 μM S1th 13 (6)
20 μM S1th 12 (5)
30 μM S1th 10 (7)
30 μM SirReal2 [Sirt2 inhibitor, synthesized according to (62)]

dependent variables

Oxidative stress induced using 10 μM Camptothecin 2 h
H2AX and γH2AX protein levels (measured by western blot)

control variables

Sirt1 WT and KO MEFS cells
48 h treatment duration

controls

Positive control: Not specified
Negative control: Sirt1 KO MEFS cells

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