K. Seifert et al. / Bioorg. Med. Chem. 20 (2012) 6465–6481
6481
Max ꢀ Min
reader (Tecan, Salzburg, Austria) and Magellan standard software.
Each assay was replicated three times by independent
experiments.
y ¼ Min þ
p
x
1 þ ðEC
Þ
50
Data were first normalized so that individual dose-response
curves converged to 0 and 100. Subsequently, these normalized
data were used to calculate the EC50 and corresponding SE and con-
fidence intervals based on 2–4 replicates. Examples for the calcula-
tion of concentration-response curves are shown for SAG (10a) and
10c. Calculations were performed using the software jmp (SAS
Institute Inc., Cary, NC). EC50 with non-overlapping confidence
intervals (p <0.05) were considered as significantly different.
Acknowledgments
We thank Dr. Stefan Scholz (UFZ-Helmholtz Centre for Environ-
mental Research, Department of Bioanalytical Ecotoxicology, Leip-
zig) for the ability to conduct the cell-based assay for Hh pathway
activation in his facilities, and Dr. Lothar Hennig for recording NMR
spectra. This work was partly financially supported by the TRM-
Translational Centre of Regenerative Medicine Leipzig.
3.2.3. Cell viability assay
The cytotoxicity was investigated in human umbilical vein
endothelial (HUVE) cells (PromoCell) by incubation with cell prolif-
eration reagent WST-1 (Roche, Mannheim, Germany). Briefly, 7500
Supplementary data
Supplementary data associated with this article can be found, in
cells per well (Greiner Bio-one, 96 well microplate, white,
bottom) were exposed to different compound concentrations
(100 l/well, containing less than 0.5% DMSO (v/v)) for 24 h before
lclear
l
References and notes
adding the WST-1 reagent. After 24 h of incubation absorbance
(440 nm and 650 nm) was measured according to the test protocol.
For determination of cytotoxicity, absorbance difference was plot-
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3.2.6. ELISA
Primary fibroblasts of foreskin tissue were placed on plastic cul-
ture dishes in Dulbecco’s modified Eagle’s medium (Gibco) con-
taining 10% fetal bovine serum (FBS) for 24 h. After removing of
nonadherent cells by washing with PBS (Gibco) cells were reseed
on 24 well plates (14000 cells per well) in a final volume of
500
l
l per well. After 72 h the medium was replaced and the cells
l per
were treated by different compound concentration (500
l
well, containing less than 0.1% DMSO (v/v)). After incubation for
48 h at 37 °C and 5% CO2 cell supernatants were taken and stored
at ꢀ20 °C.
VEGF165-concentration in conditioned media from compound-
treated cells was compared with vehicle-treated cells. VEGF165
was determined per manufacturer’s instructions using the Quanti-
kine human VEGF-ELISA kit (R&D Systems, Wiesbaden, Germany).
The concentrations were determined using a 96 well ELISA plate