These proliferating cells were selleckchem Idelalisib treated with RSV 0. 1 and 25 uM. These two doses represent the optimal concentrations to induce ef fects Inhibitors,Modulators,Libraries on differentiation process without any significant toxicity for cells. This observation was validated by our growth curve and cell viability test. According to RSV half life, medium was changed every 8 hours. Mouse myoblast C2C12 immortalized cell line is a subclone of C2 myoblasts, which spontaneously fuse and differentiate into multinucleated myotubes as a result of both the achievement of myoblast confluence and the removal of the serum growth factors. Figure 1B explains experimental study design in each phase of the protocol, with cell confluence percentage, treatments start time and duration.

RSV action was evaluated by Real Time PCR, Western Blot and Immunofluorescence analysis during prolifera tion phase and in the induction, progression and termin ation of myogenesis. RSV effects on hypertrophy process were also studied. Growth curve and cell viability test To study RSV action on C2C12 myoblast proliferation, Inhibitors,Modulators,Libraries we performed growth curve assay as described. C2C12 myoblasts were plated in 60 mm 15 mm cul ture dishes at 40% confluence and grown in GM with or without RSV. Medium was changed every 24 h and the experiment lasted until control cells achieved 70% of confluence. Every day, the cells were trypsinized and stained with trypan blue. Both viable and non viable cells were counted using a hemacytometer. The total cell count average values for each single day were used to plot a growth curve for myoblasts treated with RSV and control.

Cell viability was calculated by dividing the non stained vi able cell count by the total cell count. In addition, every day morphological changes were examined. Real Time Inhibitors,Modulators,Libraries PCR array analysis RT2 PCR Array plates produced by SABiosciences were utilized to simultaneously analyze the expression levels of a panel of genes. We studied the following genes expression during pro liferation phase Cyclin A2, Cyclin B1, Cyclin Inhibitors,Modulators,Libraries C, Cyclin D1, Cyclin E1 and Cyclin F, using Mouse Cell Cycle RT2 Profiler PCR Array, as described. Total RNA was isolated from C2C12 using the RNeasy Plus Mini Qiagen Kit. Total RNA was reverse transcribed using RT2 First Strand Kit. The reverse transcripts were used as templates for analysis of gene expression level using RT2 PCR Arrays plates according to the manufacturers instructions.

Each sample Inhibitors,Modulators,Libraries was run in triplicate. The expression level of the housekeeping genes chosen for normalization in the thresh old cycle for each experimental conditions and then the fold change for each gene from treated group compared to the control group, was calculated. If the Ct is greater than 1, the result may be reported as a fold up regulation. If the Ct is less than www.selleckchem.com/products/Lenalidomide.html 1, the result may be reported as a fold down regulation.