Fig. 6.

Expression and activity of ADAM10 in contact inhibited SH-SY5Y cells and murine dermal fibroblasts. a Senescence was induced in SH-SY5Y cells by contact inhibition (CI) and detected by fluorescence-based measurement of senescence-associated beta-galactosidase activity. mRNA levels of ADAM10 (c), TBX2 and T-box family members TBX3 and TBX21 (b) were analyzed by real-time RT-PCR. Obtained values were normalized to those of GAPDH and set in relation to control cells (low plating density). d APPs-alpha, ADAM10, and TBX2 protein level were determined by western blot using 20 µg of protein from cell lysates and the respective antibodies. Both, the proform and mature ADAM10 were analyzed separately and set in relation to values from control cells. Secreted proteins of respective supernatants were subjected to western blot analysis and ADAM10-dependent soluble fragment of APP, APPs-alpha, was detected with a specific N-terminal antibody. GAPDH was used as loading control. Results are displayed as mean ± SD of two independent experiments performed in triplicate (ns p > 0.05, *p < 0.05, ***p < 0.001, Unpaired Student’s t test). e Primary murine dermal fibroblasts from four independent donor mice were co-transfected with Tbx2-expression vector and murine Adam10 promoter reporter. Luciferase-activity as a reporter for Adam10 transcriptional activity was measured and normalized to empty control vector transfected cells (set to 100%) (**p < 0.01, Unpaired Student’s t test). f Murine dermal fibroblasts were grown in two different densities (control and contact inhibited) and Adam10 as well as Tbx2 mRNA level quantified via real time RT PCR. 18SrRNA levels served for normalization and control values were set to 1 (*p < 0.05, Unpaired Student’s t test)