Determination of factors affecting overall survival.

<p>Patients overall survival analyses were made between two groups: (A) patients with or without mutations in <i>NLRP</i> genes; (B) patients with or without mutations in <i>TP53</i> genes; (C) patients with tumors that harbored <i>TLR</i> mutations and those without <i>TLR</i> mutations; (D) patients with or without HPV infections; (E) patients with low stage (stage II) or advanced stage (stage III and beyond) tumors; (F) patients of less or more than 56 years old; (G) patients who received no adjuvant therapy, radiotherapy alone or both chemotherapy and radiotherapy.</p

Identification of <i>NLRP</i> mutations in FOM HNSCC.

<p>Mutations in four <i>NLRP</i> genes were identified in FOM HNSCC patients. Black triangles indicate novel mutations identified in this study. Gray triangles represent reported mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.</p

Mutation rates of FOM HNSCC.

<p>(A) Numbers of missense and nonsense mutations were compared between patients with FOM or non-FOM HNSCC. (B) Mutation rates were compared between patients with FOM or non-FOM HNSCC.</p

Demographic Profile of Study Subjects with Primary Conventional HNSCC (PC-SCC).

<p>Contingency tables were analyzed by Fisher's exact test. Survival distributions were analyzed by Log Rank test. Notes:</p>†<p>Typical number of alcohol drinks in 2 week period, §Mann-Whitney U test,</p>∥<p>Fisher's exact test,</p>¶<p>Log rank test,</p>*<p>Comparing patients with or without mutations in <i>NLRP</i> genes.</p

Identification of <i>NLRP</i> mutations in primary HNSCC.

<p>Novel mutations were identified in 10 <i>NLRP</i> genes in HNSCC. Most mutations were involving chromosomal locations 11p15.4 and 19q13.42-19q13.43.</p

Mutation rates comparisons.

<p>(A) Numbers of missense and nonsense mutations were compared between tumors with or without <i>NLRP</i> mutations. (B) Numbers of missense and nonsense mutations were compared between tumors with or without <i>TP53</i> mutations. (C) Numbers of missense and nonsense mutations were compared between tumors with or without <i>TLR</i> genes mutations. (D) Mutation rates [shown as mutations per million base (MB) pairs] were compared between tumors with or without <i>NLRP</i> mutations. (E) Mutation rates [shown as mutations per million base (MB) pairs] were compared between tumors with or without <i>TP53</i> mutations. (F) Mutation rates [shown as mutations per million base (MB) pairs] were compared between tumors with or without <i>TLR</i> genes mutations. P value less than 0.05 was considered significant.</p

STAT3 decoy inhibits HUVEC tubule formation.

<p>(A) HUVECs were transfected with 100 nM of STAT3 decoy oligonucleotide or 100 nM of STAT3 mutant control oligonucleotide. Twenty four hours later 15,000 cells were plated on top of Matrigel in a 96-well plate. After 24 additional hours the plates were assessed for tubule formation. The tubule formation was the quantified by counting the total number of (B) nodes and (C) tubules per field. Stat3 decoy oligonucleotide decreased both node and tubule number compared to the STAT3 mutant (*, ** p<0.05). All of the experiments were performed in triplicates.</p

Effects of STAT3 decoy oligonucleotide on the proliferation, apoptosis, and migration of HUVECs and HDMECs<i>in vitro</i>.

<p>(A) HUVECs and (B) HDMECs were transfected with the STAT3 decoy oligonucleotide or mutant control oligonucleotide at 0 nM to 1000 nM. Seventy two hours later the cells were assessed for proliferation using MTT assay. Results are shown in percent kill relative to the mutant control oligonucleotide and the experiments were performed in triplicate. (C) HUVECs were transfected with the either STAT3 decoy oligonucleotide (0 nM to 100 nM), the mutant control oligonucleotide (100 nM), vehicle, or cisplatin (100 µM). Twenty four hours later the cells were analyzed for apoptosis using Annexin V staining and flow cytometry. The experiments were performed in triplicate (* p<0.05). (D) HUVECs were transfected with 100 nM of STAT3 decoy oligonucleotide or 100 nM of STAT3 of mutant control oligonucleotide, plated inTranswell8uM pore inserts, and extent of migration was ascertained 24 hrs later. STAT3 decoy oligonucleotide decreased the migration of HUVEC compared with the mutant control oligonucleotide (* p<0.05). All of the experiments were performed in triplicate.</p

STAT3 decoy inhibits net migration of HUVECs during <i>in vitro</i> tubule formation.

<p>HUVECs were transfected with 100× every ten minutes for the following 24 hours in eight different fields. (A) The decoy was FAM-tagged (green) in order to ensure positive transfection. (B) Representative images from various time points from the course of the experiment.The live cell images were quantified for three distinct parameters: (C) mean distance traveled,(D) mean velocity, and (E) distance from origin. The distance from origin was significantly decreasedby STAT3 decoy oligonucleotide compared to the STAT3 mutant oligonucleotide (** p<0.05). All of the experiments were performed in triplicates.</p

STAT3 decoy decreases microvessel density <i>in vivo</i> by acting directly on the endothelial cells.

<p>Ploy-L-lactic acid scaffolds were seeded with HUVECs and then implanted on the flank of nude athymic mice. The mice were the treated with either the STAT3 decoy oligonucleotide or the STAT3 mutant control via peri-scaffold injection for 15 days. (A&B)) Representative images from CD31 staining of scaffolds treated with either STAT3 decoy of STAT3 mutant control showing decreased microvessel density with STAT3 decoy oligonucleotide. (C) Quantification of microvessel density in both treatment groupsshowed decreased microvessel density in STAT3 decoy oligonucleotide treated group (*p<0.05).</p