CSC vapor induces Survival Proteins.

<p>(a) Cellular lysates were obtained from the indicated cell lines and subjected to Western Blot analysis using the indicated antibodies. β-actin was used as a control. (b) The indicated cell lines were treated with 3 µg/ml cisplatin for 48 h. Mitochondrial and cytoplasmic total cellular lysates were subjected to Western Blot analysis using anti-cytochrome c antibody. Protein extracts in each lane are as indicated. Immunodetection of â-actin and COX II were done to assure that the transfected proteins were in cytoplasm and mitochondria respectively.</p

CSC Vapor Induces Cisplatin Resistance In Xenograft Model.

<p>(a) Subcutaneous xenografts were generated in of 5- to 6-week-old female athymic <i>nu/nu</i> mice with SCaBER or SCaBER-6M cells. One week following tumor cell inoculation, mice were treated with either PBS or cisplatin (3 mg/Kg/dose). The mean tumor volume was calculated. * indicates <i>P</i><0.01 (b) One representative mouse from each group was displayed.</p

CSC vapor decreased the expression of AK3 and increased cisplatin resistance.

<p>(a) Western blot analysis was performed using anti-<i>AK3</i> and anti-<i>AK2</i> antiserum. Protein extracts in each lane are as indicated. Even loading was confirmed by re-probing the membrane with β-Actin antibody. HUC1-6M or SCaBER-6M indicates the cells chronically exposed to CSC vapor. HUC1-0.1%6M are SV-HUC-1 cells exposed to 0.1% CSC for 6 months. SV-HUC-1 and HUC1-6M (b), SCaBER and SCaBER-6M (c) were treated with 0 to 5 µg/ml cisplatin for 48 h, or with 3 µg/ml cisplatin for 24, 48 and 72 h, in the presence and absence of AK3 as indicated. (d) Colony formation assays were performed with SCaBER and SCaBER-6M in the presence and absence of AK3 as indicated. Data were expressed as mean ±SD. * indicates <i>P</i><0.05 (e) SV-HUC-1 and SCaBER cells were transfected with AK3 RNAi for 48 h and Western blot analysis was performed using anti-AK3 antiserum. (f) SCaBER cells were transfected with AK3 RNAi and 48 h after transfection the cells were treated with 0 to 5 µg/ml cisplatin for 48 h and cellular survival was assessed. Error bars represent standard deviation of three experiments.</p

ROS Production by AK3 in the Presence of Cisplatin.

<p>ROS production was measured in parental SV-HUC-1 and SCaBER cells or cells exposed to CSC vapor for 6 months using DCFH-DA staining. The cells were transfected with or without the AK3 expression plasmid and treated with or without cisplatin for 48 h before staining. ROS generation in SV-HUC-1 and HUC1-6M (a–c) and SCaBER and SCaBER-6M (d–f) cells with overexpression of AK3 with and without cisplatin. The mean fluorescence density was calculated and data was plotted as mean ± SD (g,h).</p

AK3 Induced Release of Lactate Dehyderogenase upon Treatment of Cisplatin.

<p>LDH release in SV-HUC-1 (a) and SCaBER (b) cells exposed to CSC vapor in presence of AK3 followed by cisplatin treatment for 48 h. The data are presented as means ± SD. All experiments were performed in triplicate. Statistical significance is as indicated with * indicates <i>P</i><0.05 and ** indicates <i>P</i><0.01</p

Activation of Apoptosis in the Presence of AK3 and Cisplatin.

<p>Apoptosis was measured in the SV-HUC-1 and HUC1-6M cells using annexin/PI staining. The cells were transfected with or without AK3 expression plasmid and treated with or without cisplatin for 48 h as indicated.</p

ΔΨm Alteration by AK3 in the Presence of Cisplatin.

<p>Mitochondrial membrane potential was measured in SV-HUC1, HUC1-6M (a), SCaBER and SCaBER-6M (b) cells transfected with AK3 or control vector, followed by treatment with cisplatin for 48 h. Cells were stained with JC-1 reagent and cell fluorescence was measured on a flow cytometer using Fl1 and Fl2 channels. Increase of red fluorescence indicates hyper-polarization of mitochondrial membrane potential (ÄΨm). * indicates <i>P</i><0.05 and ** indicates <i>P</i><0.01.</p

Supplementary Table 1 from Inhibition of TGF-β Enhances the <i>In Vivo</i> Antitumor Efficacy of EGF Receptor–Targeted Therapy

PDF file - 256K, Clinical characteristics of patients with HNSCC</p

Supplementary Figure 1 from Inhibition of TGF-β Enhances the <i>In Vivo</i> Antitumor Efficacy of EGF Receptor–Targeted Therapy

PDF file - 191K, Tumor HPV status and serum TGF-beta1 levels of HNSCC patients</p

Supplementary Table 2 from Inhibition of TGF-β Enhances the <i>In Vivo</i> Antitumor Efficacy of EGF Receptor–Targeted Therapy

PDF file - 70K, A, Serum levels of the indicated cytokines in patients with HNSCC and pleomorphic adenoma (non-cancer control). B, Comparison of serum TGF-beta1 levels in non-cancer controls and patients with HNSCC at the time of new diagnosis and at the time of recurrence. C, Comparison of serum levels of TGF-beta1 in patients with HPV-negative HNSCC at the time of diagnosis, and at two time points after the completion of treatment</p