of 26103 and 56103 cells/well respectively. At the completion of incubation, cell proliferation was assessed by adding 10 ml of MTT labeling reagent into each well and incubating at 37uC for 4–6 hrs (4 hrs for tumor cells and 6 hrs for HDMEC). Reaction was stopped by adding solubilization solution and incubating the plates at 37uC overnight. The plates were read on a microplate reader (Sectramax 190, Molecular Devices Corp., Sunnyvale, CA) at a wavelength of 590 nm. The percentage cell proliferation for each group was calculated by adjusting the control group to 100%. For Xcelligence system proliferation assay, 100 ml of media (DMEM for tumor cells and EGM for EC) containing 2% FBS was added to the wells. After 1 hr of equilibration with media, 100 ml of cell suspension (3,000 tumor cells or 5,000 endothelial cells) was added to each well. Cell proliferation was monitored and expressed as percentage cell proliferation. Motility Assay Tumor and endothelial cell motility were examined by scratch assay [66] and Xcelligence system [67]. For scratch motility assay, a fine groove was made using a sterile pipette tip in about 90% confluent cells. The migration of cells was monitored microscop- ically using Nikon Eclipse Ti microscope with DS-Fi1 camera. For Xcelligence system migration assay, 160 ml of media (DMEM for tumor cells and EGM for EC) containing 10% FBS was added to the lower chambers. Upper chamber (sensor surface facing down) was then carefully assembled on top of lower chamber and 50 ml of serum free media was added to the wells. After 1 hr of equilibration with media, 100 ml of cell suspension (50,000 cells/ well in serum free media) was added to each well. Cell migration to lower chamber was monitored and expressed as cell migration index at 24 hrs. Tumor Cell Colony Formation Assay Tumor cells (5x103) were plated in 60 cm petri dishes and cultured at 37uC. After 14 days of culture, colonies were stained with crystal violet (0.005%) for 20 minutes and photographed. Alpha Innotech (San Leandro, CA) imaging software was used to quantify tumor cell colony numbers. ELISA Tumor cells were cultured in 6-well plates till they are 80% confluent. Cells were washed and fresh media was added. After 24 hours, culture supernatants were collected and cell number in each well was counted. VEGF levels in culture supernatants were measured using Quantikine human ELISA kits (R&D Systems, Minneapolis, MN) as per manufacturer’s instructions and normalized to 1x106 cells. Western Blot Analysis Whole cell lysates were separated by 4–12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred onto PVDF membranes. Nonspecific binding was blocked by incubating the blots with 3% BSA in Tris buffered saline containing 0.1% Tween-20 (TBST) for 1 hr at room temperature (RT). The blots were then incubated with primary antibody in TBST +3% BSA at 4uC overnight. After washing with TBST, the blots were incubated with horseradish peroxidase-conjugated sheep anti- mouse IgG (1:5,000) or with donkey anti-rabbit IgG (1:5,000) for 1 hr at RT. An ECL-plus detection system (GE Healthcare, Piscataway, NJ) was used to detect specific protein bands. Protein loading in all the experiments was normalized by stripping the blots and then re-probing with anti-tubulin antibody. Alpha miR-34a Inhibits HNSCC Tumor Growth Innotech (San Leandro, CA) imaging software was used to quantify Western blot bands. Matrigel in vitro Endothelial Tube Formation Assay Endothelial cell tube formation was performed on Matrigel coated chamber slides as described previously [68]. Each assay was photographed (Nikon Eclipse Ti microscope with DS-Fi1 camera) at 40x magnification and total area occupied by endothelial cell derived tubes in each chamber was calculated using software (NIS- Elements-Basic Research, Nikon, Melville, NY) and expressed as an angiogenic score. Xenograft Tumor Model Tumor cells (1 x106) were mixed with 100 ml of Matrigel and injected subcutaneously in the flanks of SCID mice (n = 5) as described previously [40]. Tumor volume measurements began on day 3 and continued twice a week until the end of the study. The length and width of the tumors were measured using a digital caliper and tumor volumes were calculated using the formula, volume (mm3) = L6W2/2 (length L, mm; width W, mm). After 18 days, tumors samples were carefully removed. A piece of each of the primary tumor was used to extract RNA to confirm the presence of miR-34a by real-time PCR. Rest of the tumors were fixed with 4% paraformaldehyde and then processed to form paraffin embedded tissue blocks for immunohistochemistry. Tumor sections were stained for angiogenesis (CD31) as described previously [68]. Microvessel density was calculated by counting 5 random high power fields (200x). Statistical Analysis Data from all the experiments are expressed as mean 6 SEM from a minimum of 3 independent experiments. The miR-34a expression in patient samples and head and neck cancer cell lines (Figure 1) was analyzed by Mann-Whitney test. To examine the inverse relationship between miR-34a and survivin expression in patient samples (Figure 4F), miR-34a levels for each tumor sample was plotted against the inverse of survivin levels and Pearson correlation coefficient was calculated. The rest of the data was statistically analyzed by two-way analysis of variance or Student’s t test (wherever applicable) and a p value of ,0.05 was considered significant. Supporting Information Table S1 (TIF) Figure S1 miR-34a significantly downregulates SIRT1, CDK4, E2F3a/b and survivin protein expression. UM- SCC-74A cells were transfected with miR-34a or SC. Seventy two hrs after transfection whole cell lysate was prepared and Western blotted using antibody specific for SIRT1 (top panel) or CDK4 (second panel) or E2F3a/b (third panel) or survivin (fourth panel). Equal protein loading in Western blotting was verified by stripping the blots and reprobing with tubulin antibody. (TIF) Figure S2 miR-34a significantly inhibits tumor cell proliferation. UM-SCC-74A cells were transfected with miR- 34a or SC. Seventy two hrs after transfection, cells were plated in 96 well plates and cell proliferation was examined at different time points using MTT assay. (TIF) Head and neck cancer cell line characteristics. PLoS ONE | www.plosone.org 11 May 2012 | Volume 7 | Issue 5 | e37601