Journal: Oncotarget

Article Title: Cigarette smoke and decreased DNA repair by Xeroderma Pigmentosum Group C use a double hit mechanism for epithelial cell lung carcinogenesis

doi: 10.18632/oncotarget.28724

Figure Lengend Snippet: Clonogenic survival assays were conducted on XPC knockdown (shXPC) cells in comparison to the control (shCtrl) under varying concentrations of CS extract (CSE) for ( A ) A549, ( B ) H1299 and ( C ) H520 non-small cell lung cancer (NSCLC) cell lines. The survival data is presented as individual points with error bars indicating standard error of the mean (±SEM) and fitted to a best-fit four-parameter logistic curve. Statistical differences in CSE survival were assessed for each shXPC cell line compared to shCtrl using a two-way ANOVA with repeated measures with no significant differences were observed in the clonogenic survival of the human NSCLC cell lines.

Article Snippet: Relative quantification in real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan Gene Expression Master Mix and primers (Applied Biosystems) for XPC (Hs01104206_m1) and an endogenous control (GAPDH, 4333764F) as published [ ].

Techniques: Knockdown, Comparison, Control

Journal: Oncotarget

Article Title: Cigarette smoke and decreased DNA repair by Xeroderma Pigmentosum Group C use a double hit mechanism for epithelial cell lung carcinogenesis

doi: 10.18632/oncotarget.28724

Figure Lengend Snippet: The impact of XPC on cigarette smoke-induced apoptosis is shown in H1299 ( A – D ) H520 ( E – H ) and A549 ( I – L ) NSCLC cell lines. Flow cytometry shows Annexin V-PI stain and quantification of early and late apoptotic cells in H1299 (A, B), H520 (E, F) and A549 (I, J) cells. Solid bars = Annexin V+ (early apoptosis). Striped bars = Annexin-V-PI + (late apoptosis). Confirmation of apoptosis by Western blot for activated PARP, Caspase 9, and Caspase 3 are shown in (C) H1299, (G) H520 and (K) A549 NSCLC cell lines. Quantification of densitometry is shown for H1299 (D), H520 (H) and A549 (L). Data are presented as mean ± SEM from 3–4 independent experiments. Statistical significance was determined using 2-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Relative quantification in real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan Gene Expression Master Mix and primers (Applied Biosystems) for XPC (Hs01104206_m1) and an endogenous control (GAPDH, 4333764F) as published [ ].

Techniques: Flow Cytometry, Staining, Western Blot

Journal: Oncotarget

Article Title: Cigarette smoke and decreased DNA repair by Xeroderma Pigmentosum Group C use a double hit mechanism for epithelial cell lung carcinogenesis

doi: 10.18632/oncotarget.28724

Figure Lengend Snippet: Total DNA damage measured following 24 hours of exposure to cigarette smoke extract (CS) or filtered air (AC) in ( A ) benign epithelial cells (Beas-2B), ( B ) A549 lung adenocarcinoma cell line, ( C ) H1299 lung adenocarcinoma cell line and ( D ) H520 lung adenocarcinoma cell line modified by XPC knock-down (shXPC) or scramble control (shCtrl). Note increased DNA damage correlates to increasing CS concentrations and is further increased by shXPC compared to shCtrl in all cell lines. Mean +/− SD from 3 separate experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA.

Article Snippet: Relative quantification in real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan Gene Expression Master Mix and primers (Applied Biosystems) for XPC (Hs01104206_m1) and an endogenous control (GAPDH, 4333764F) as published [ ].

Techniques: Modification, Knockdown, Control

Journal: Oncotarget

Article Title: Cigarette smoke and decreased DNA repair by Xeroderma Pigmentosum Group C use a double hit mechanism for epithelial cell lung carcinogenesis

doi: 10.18632/oncotarget.28724

Figure Lengend Snippet: Analysis of 8-hydroxy-2’-deoxyguanosine (8-OHdG) adducts following 24-hour exposure to cigarette smoke extract (CS, +) or filtered air (−) using FLARE Comet Assay and human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1) in ( A ) Benign bronchial epithelial cells (Beas-2B) modified by XPC knock-down (shXPC) or scramble control (shCtrl), ( B ) H1299 NSCLC cells modified by shXPC compared to shCtrl and ( C ) A549 NSCLC cells modified by shXPC compared to shCtrl. Note the amount of oxidative DNA damage increases with CS concentration and exhibits a more pronounced impact in shXPC compared to shCtrl in all cell lines. Mean +/− SD from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-way ANOVA.

Article Snippet: Relative quantification in real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan Gene Expression Master Mix and primers (Applied Biosystems) for XPC (Hs01104206_m1) and an endogenous control (GAPDH, 4333764F) as published [ ].

Techniques: Single Cell Gel Electrophoresis, Modification, Knockdown, Control, Concentration Assay

Journal: Oncotarget

Article Title: Cigarette smoke and decreased DNA repair by Xeroderma Pigmentosum Group C use a double hit mechanism for epithelial cell lung carcinogenesis

doi: 10.18632/oncotarget.28724

Figure Lengend Snippet: Representative DAPI images of ( A ) micronuclei (white arrow) and ( B ) nuclear aberrancies (nuclear blebs or bridges, red arrow) in Beas-2B cells exposed to CSE. ( C , D ) Quantification of % Beas-2B cells with micronuclei (C) and % cells with nuclear aberrancies (D). Results are also shown for H1299 ( E , F ) and A549 ( G , H ). Abbreviations: shXPC: lentiviral XPC knock-down. shCtrl: scrambled shRNA control. Data are presented as mean ± SEM. Statistical significance was determined using Two-Way ANOVA. ** p < 0.01; *** p < 0.001 by 2-way ANOVA.

Article Snippet: Relative quantification in real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan Gene Expression Master Mix and primers (Applied Biosystems) for XPC (Hs01104206_m1) and an endogenous control (GAPDH, 4333764F) as published [ ].

Techniques: Knockdown, shRNA, Control

Journal: Oncotarget

Article Title: Cigarette smoke and decreased DNA repair by Xeroderma Pigmentosum Group C use a double hit mechanism for epithelial cell lung carcinogenesis

doi: 10.18632/oncotarget.28724

Figure Lengend Snippet: Relative NER efficiency of UV-modified plasmid (pMAX-GFP, UV) or unmodified plasmid (C) in cells treated with increasing concentrations of CSE or air control (AC). ( A ) %NER in bronchial epithelial cells (Beas-2B) is higher at baseline and decreases significantly after treatment with CSE. ( B ) %NER in human A549 NSCLC cell line is decreased at baseline and not significantly altered by CSE. Shown as mean +/− standard deviation from 3 separate experiments. Abbreviation: UV: ultraviolet. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-Way ANOVA. ( C ) XPC mRNA expression by RT-qPCR is decreased in Beas-2B cells exposed to CSE for 24 hours. Statistical analysis by one-way ANOVA using dCt values.

Article Snippet: Relative quantification in real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan Gene Expression Master Mix and primers (Applied Biosystems) for XPC (Hs01104206_m1) and an endogenous control (GAPDH, 4333764F) as published [ ].

Techniques: Modification, Plasmid Preparation, Control, Standard Deviation, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Cigarette smoke and decreased DNA repair by Xeroderma Pigmentosum Group C use a double hit mechanism for epithelial cell lung carcinogenesis

doi: 10.18632/oncotarget.28724

Figure Lengend Snippet: ( A ) XPC mRNA expression decreased in unmatched samples from lung adenocarcinoma (AdenoCA) compared to benign lung from the TCGA database. ( B ) Decreased XPC mRNA expression in frozen lung adenocarcinoma compared to non-cancerous (benign) lung. Box plot with median and 25–75%, whiskers at 10% and 95%. ( C ) Ratio of XPC mRNA expression in lung adenocarcinoma to subject matched benign lung resected at the time of surgery, individual subjects shown on Y-axis. ( D ) XPC mRNA expression decreased in unmatched samples from the TCGA database of lung squamous cell carcinoma (LUSC) and benign lung. ( E ) Decreased median XPC mRNA expression in frozen lung squamous cell carcinoma compared to benign lung. Box plot with median and 25–75%, whiskers at 10% and 95%. ( F ) Ratio of XPC mRNA expression in lung squamous cell carcinoma to subject-matched benign lung resected at the time of surgery, individual subjects shown on Y-axis. *** p < 0.001 by one way ANOVA.

Article Snippet: Relative quantification in real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan Gene Expression Master Mix and primers (Applied Biosystems) for XPC (Hs01104206_m1) and an endogenous control (GAPDH, 4333764F) as published [ ].

Techniques: Expressing