tween for 5 minutes, twice in TBS without tween, dried, and scanned with the Li cor Pimecrolimus Odyssey at 21 lm resolution and high quality. Blots were analyzed by use of Li cor Odyssey analysis software . Circles were applied to the band on the scanned image, and the net intensity was calculated by subtracting the background intensity from the trimmed mean intensity of each sample. The net intensity was divided by the net intensity of tubulin to control for lysate protein concentration. Biologic replicates were averaged and graphed, with error bars showing the standard error of each sample. Samples were normalized with the vehicle treated control lysates set to 1 and treatments shown as a fraction of the control for each set of treatments. Cell Growth Inhibition Assay with MTT.
MTT assays were used to determine cell viability. Briefly, cells were seeded at 1000 cells/well on 96 well plates, incubated for 24 hours, maintained in serum free conditions for 24 hours, and then treated with enzastaurin or rapamycin in serum containing Streptozocin clinical trial medium for 72 hours at 37”C. After drug treatment, MTT Labeling Reagent was added to each well and incubated for 4 hours at 37”C before the solubilization solution was added. Cell viability was quantified by scanning absorbance at 550 nm in a microplate reader . Experiments were performed in quadruplicates for each drug concentration. Determination of Synergy. Isobolograms were plotted by use of the Calcusyn software package and synergy calculated on the basis of the median effect equation as described by Chou and Talalay.
11 Apoptosis Analysis by Flow Cytometry. Induction of apoptosis was assessed by flow cytometry using Annexin V FITC Apoptosis Detection Kit I . Cells were seeded in 100 mm dishes and treated in serum containing medium Streptozocin structure with 50 nM of rapamycin or 10 lM of enzastaurin at various time points. After treatment, cells were trypsinized, washed twice with PBS, and then resuspended in 100 lL Annexin V binding buffer. Cells were incubated with 5 lL of Annexin V FITC and 5 lL of propidium iodide at room temperature for 15 minutes in the dark and then analyzed by BD FACSCanto flow cytometer . The data analysis was performed with Flowjo software . Results were compared by use of 2 sided paired t test. Tumor Xenografts. Female athymic nude mice at 6 to 7 weeks of age were maintained in a pathogen free animal facility in accordance with the University of Chicago Animal Care and Use Committee.
Mice received standard laboratory rodent food and water as desired. All handling procedures were conducted in a laminar flow biosafety hood. CAL27 cells were used to induce SCCHN tumor xenografts. Cells were harvested, washed, resuspended in PBS, and 1 107 cells were injected subcutaneously into the right hind limb of nude mice. For tumor growth Streptozocin solubility analysis tumor size was measured every other day with a vernier caliper. Tumor volumes were calculated with the formula V ¼ 0.52 LW2, where L and W represent the length and the width of the tumor. The wealth inequalities animals were monitored 2 times per week for body weight. The drug treatment was initiated when mean volumes reached 100 to 150 mm3. Tumor bearing animals were randomly divided into 4 groups of 10 animals. At the end of the experiment, there were 8 mice left .