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

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

Pattern of mtDNA mutation in patient 2.

<p>The patient was operated on 2005 for surgical removal of tumor from the right upper lobe. Five bronchoscopically abnormal airway mucosal biopsies were obtained from main carina (M1), right lower lobe (M2 and M3) and right upper lobe (M3 and M4) surrounding the tumors as depicted (Panel A–E). The mucosal biopsies exhibited a range of 2–5 mtDNA mutations as represented in different color (Panel A–E). Identical mutations are shown in same color in different biopsies and the tumor. A different color code was also used for each mucosa to indicate the clonal progression of the lesions with accumulated mtDNA mutations. Representative histological photomicrograph of the tumor and normal mucosa are shown in Panel A and D. The tumor exhibited 9 mtDNA mutations (Represented in the Bottom rectangle) including all the 8 mutations exhibited by the five mucosal biopsies (Matched color). An additional mtDNA mutation (<i>T7001C</i>) detected in the tumor is underlined in black in the bottom rectangle. Tumor was encircled to indicate its development from the clonal mucosal patches. M: Mucosa.</p

Pattern of mtDNA mutation in patient 3.

1<p>Revised Cambridge Reference Sequence;</p>2<p>Mucosal biopsies from 5 different regions were pooled together because of small sample size;</p>3<p>Tumor adjacent histologically normal margin.</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

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

MtDNA content in the lung cancer patients.

<p>mtDNA content was measured by multiplex real-time PCR using nuclear encoded β-actin and mitochondria encoded COI gene. A ratio of COI/β -actin corresponds to the fold difference compared to control. MtDNA content increased significantly (P<0.001) in the mucosal biopsies and corresponding tumors compared to the normal control in both the patients as indicated. N: Normal lymph node; M: Mucosa; T: Tumor. *P value <0.05 compared to normal lymph node.</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

Pattern of somatic mtDNA mutation in the 8 lung cancer patients.

1<p>RCRS: Revised Cambridge Reference Sequence;</p>2<p>Normal Lymphocytes.</p

The pattern of germ line mtDNA sequence variants in the lung cancer patients.

1<p>Revised Cambridge Reference Sequence;</p>2<p>Matched normal lymphocytes;</p>3<p>R: Reported; N: Novel as per Human Mitochondrial Database and relevant literature.</p