Afterwards, 1 ug of total RNA was reverse transcribed using the First Strand cDNA synthesis kit according to manufacturer s instructions. The cDNA equivalent of 5 ng RNA was used for amplification inhibitor Trichostatin A in 384 well microtiter plates in a TaqMAN ABI7900HT cycler in a final re action volume of 10 ul containing 5 ul SyberGreen uni versal PCR Master Mi and 6 uM primer mi . 6 uM of mouse Beta 2 microglobulin and human GAPDH primer mi were used as a reference gene. Cycle threshold values for individual reactions were determined using ABI Prism SDS 2. 2 data processing software. To determine the differences in e pression, the CT values were normalized to reference gene using the Ct method, normalizing for the e pression of the reference gene and related to the control treatment. All cDNA samples were amplified in duplicate.
ELISA ADSC conditioned medium was collected and filtered through 0. 2 um filter to remove any residual debris. To quantify the IL 6 production by ADSC, collected media were assessed by enzyme linked immunosorbent assay according to manufacturer s protocol. Absorbance values for individual reactions were determined using VersaMa Microplate Reader with Softma Pro 3. 0 data processing software. To assure statistically relevant data, samples were run in trip licate from three independent donors. Immunoblot analysis Confluent rnCM or HL 1 cardiomyocyte cultures were serum starved overnight. Subsequently, 50 uM Stattic or 10 uM UO126 and solvent controls were added to HL 1 cells for 2h. Ne t, rnCM or HL 1 cultures were treated with ADSC conditioned medium for 30min.
Protein lysates from serum depleted, confluent cultures of HL 1 cells were prepared in RIPA buffer supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitors Cilengitide cocktail 2, 3. Cell lysates were run on 10% polyacrylamide electrophoresis gel and blot ted onto nitrocellulose membrane according to standard protocol. Blots were blocked in Tris buffered saline containing 5% BSA for 1 h. Subsequently, blots were incubated in TBS 1% Tween containing 5% BSA with primary antibodies to human p STAT3, STAT3, p Erk1 2, Erk1 2, diluted 1 1000, overnight. Afterwards, blots were washed and incubated with alkaline phosphatase conjugated antibodies to mouse or rabbit IgG, at the di lution 1 2000 for 1 h. NBT BCIP was used as a substrate for detection. Densitometric analysis was performed using Totallab 120.
Immunofluorescence microscopy and image analysis rnCM and HL 1 cardiomyocytes were seeded semi confluent onto polystyrene 8 chamber slides. Subsequently, cells were serum starved in serum free Claycomb Medium overnight. Afterwards, samples were stimulated with sellectchem 10 ng ml IL 6, conditioned media of ADSC and conditioned media of ADSC supplemented with IL 6 neutralizing antibody or Mock IgG as a control for 24 h.