A model of the onset of the senescence associated secretory phenotype after DNA damage induced senescence
Fig 7
Schematic overview of the experimental workflow.
Murine dermal fibroblasts (MDFs) are isolated from NEMO-floxed mice. After short expansion in cell culture these MDFs are transfected with pCAG-Cre-T2A-mRuby2 or pCAG-mRuby2, respectively. Because of mRuby2 expression, successfully transfected cells can be sorted by FACS. Cells transfected with pCAG-Cre-T2A-mRuby2 are knocked out for NEMO while pCAG-mRuby2 transfected cells are used as wildtype controls. After transfection cells are treated with 25 μM etoposide for 3 h to induce DNA damage. 24 h after treatment cell culture media is taken for ELISA measurement of secretion and cells are harvested for RNA isolation and subsequent RT-qPCR analysis.