Fig. 2.

Characterization of TBX2-mediated repression of ADAM10 gene expression. a Time-resolved measurement of ADAM10-promoter activity. SH-SY5Y cells were transiently transfected with human ADAM10-promoter reporter construct or respective promoter-less control vector (mock) together with TBX2 expression plasmid or empty vector. Promoter activity was recorded by luminescence derived from secreted Gaussia princeps luciferase. Values obtained for empty vector transfected cells were set to 100% for each time point (indicated as red dashed line). Statistical analysis was performed by using multiple t test (*p < 0.05). b TBX2 knock-down was verified by real-time RT-PCR. Detection of TBX3 mRNA levels was used for specificity control. Exemplary results are shown from one experiment performed in duplicate (Unpaired Student’s t test was performed against the respective control; (ns p > 0.05, ***p < 0.001). c Test of TBX2-knock-down specificity. TBX2 expression plasmid or empty vector were transfected together with ADAM10-promoter reporter construct in combination with TBX2 specific sRNAi, scrambled control sRNAi or water (solvent control) in SH-SY5Y cells. Cell lysates obtained after 48 h total incubation period were analyzed via luciferase-based measurement. Values were normalized to protein content of lysates. The experiments were performed three times independently in triplicate and values denote mean ± SD. d Effect of TBX2 overexpression on ADAM10 mRNA levels. Total RNA from TBX2 expression plasmid or empty vector transfected SH-SY5Y cells was extracted and subjected to real-time RT-PCR. Obtained values for ADAM10 mRNA were normalized to those of Tubulin. Bars represent mean ± SD from three independent experiments performed in duplicate. e Analysis of the effect of TBX2 overexpression on ADAM10 and full-length APP protein level. SH-SY5Y cells were transfected with TBX2 expression vector or empty vector and cell lysates (20 µg of protein per lane) were subjected to western blot method using specific antibodies for the human proteins. GAPDH was used as a loading control and TBX2 overexpression demonstrated by a TBX2 detecting antibody. e, f–i Quantitation of ADAM10 and APP/APP cleavage product protein levels in TBX2 overexpressing cells. Exemplary blots are shown in e. Obtained values for both, the proform and mature ADAM10 (f), as well as for APP CTF_alpha (h) were normalized to the values obtained for GAPDH and ratios for empty vector transfected cells were set to 100%. Obtained values for secreted APPs-alpha were normalized to total protein amount of respective cell lysate (g). Bars represent mean ± SD of four independent experiments conducted in triplicate. i Determination of A-beta levels. Conditioned medium from TBX2 overexpressing cells was subjected to ELISA measurement and protein content of cell lysates served as normalization. Bars represent mean ± SD from two independent experiments performed in quadruplicate. (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, Unpaired Student’s t test)