Radiographic film

Genomic DNA (15 μg) was digested with EcoRI (20 units) in 150 µl reaction volume, at 37 °C overnight. DNA fragments were separated on a 0.8 % agarose gel electrophoresis overnight, and transferred to nylon membranes (Hybond-N+, Amersham) by capillary transfer according to Sambrook and Russell (2001 ). DNA fragments were bound to the membrane by UV cross- linking (Southern Stratalinker 2400). cpPLRV gene (279 bp) was used as probe labeled with [α32] dCTP using the Gene Images Random Prime Labeling kit according to the manufacturer’s recommendation. The probes were hybridized with the membranes at 65 °C for 18 h. The hybridized filter was washed with 1x SSC, 0.1 % SDS (w/v) and 0.5 × SSC, 0.1 % SDS (w/v) at 65 °C for 15 min with gentle agitation. The blot was wrapped in a polyethylene sheet and exposed to radiographic film (Kodak). To detect excision events, we used as probe the nptII gene (638 bp) labeled with PCR DIG Synthesis (Roche kit) according to the manufacturer’s conditions. The probe was hybridized at 65 °C for 30 min with 15 ml of DIG Easy Hyb solution (Roche kit) at 25 ng/mL. The membrane was washed using stringent conditions and exposed to X-ray film (Kodak) at room temperature for 16 h.

Cell culture supernatants (1 ml each sample) were collected engineered skeletal muscle formed from randomly oriented or aligned scaffolds, with or without endothelialization, on days 6 and 9 of in vitro culture (n = 3 aligned scaffold day 9, n = 4 all other groups). A human-specific angiogenic cytokine proteome profiler array (R&D Systems) was utilized according to manufacturer’s instructions, to assess the relative expression of angiogenic cytokines by endothelial cells. Radiographic film (Kodak) was exposed to chemiluminescent samples for 10 min in the dark, followed by development with an automatic film processor machine. Films were scanned at 600 dots per inch with a flatbed transmission scanner and converted to gray scale in ImageJ software. Image pixels were inverted such that white pixels were given a value of 255 and black pixels a value of 0. Using the circle tool, integrated density (mean pixel value per area) was measured for each spot on the array and the identity of each cytokine was matched using the manufacturer’s provided array legend. Duplicate spots for each cytokine were averaged, and the mean values of all experimental samples (n = 3 aligned scaffold day 9, n = 4 all other groups) were determined for each cytokine and condition. The data are expressed as relative fold change compared with engineered skeletal muscle in randomly oriented scaffolds on day 6.

To evaluate the protein levels of STAT3 and Smac, mouse peritoneal cells were subjected to western blot analysis. For this case, cells were homogenized in a 500 μL RIPA buffer containing protease inhibitor cocktail by a homogenizer after incubation for 30 min at 4°C, followed by centrifugation at 12000 × g for 10 min. subsequently protein concentrations were quantified using Bradford method. Before running samples in 10% SDS polyacrilamide gel, protein samples were boiled at 95°C for 5 minutes and 20 μg of protein/ sample was loaded in each well. Afterwards, protein bands were transferred to PVDF membrane and blocked using 1% milk/TBS for 1 hour at room temperature. Desired proteins were probed in 1% milk/TBS supplemented by specific primary antibodies of STAT3 (sc-8019, 1:300), Smac (sc-136071, 1:300) and β-actin (sc-47778, 1:300) over night at 4°C. Next day, after several washing steps, specific horseradish peroxidase (HRP) conjugated secondary antibody were probed (sc-516102, 1:1000 for STAT3 and Smac and sc-2357, 1:1000 for β-actin) and incubated 2 hour at room temperature. The protein bands were visualized using a chemiluminescence detection kit (ECL, Bio-Rad) and radiographic film (Kodak, USA). The density of bands was measured by ImageJ software. β-actin was used as a loading control.

Western blot analysis was performed as described previously^{50 (link)}. Equal amount of protein samples (50 μg) were resolved on SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) membrane. The blots were blocked with PBST (PBS and 0.05% Tween 20) containing 5% nonfat dry milk for 1 hour at room temperature, and then probed with antibodies against cleaved-PARP, cleaved-caspase 9, cleaved-caspase 7, cytochrome c, Bax, Bcl-2, CHOP, XBP-1, phospho-eIF2α, pro-caspase 12, phospho-AKT, AKT, α-tubulin for 1 hour at 4 °C. After, membranes were washed with 0.1% PBST and incubated with secondary antibodies conjugated to horseradish peroxidase for 45 min. The antibody-reactive bands were revealed using enhanced chemiluminescence reagents (Amersham Biosciences, Sweden) and exposed to radiographic film (Kodak, Rochester, NY, USA).