Journal: Cancer Research

Article Title: Nuclear Receptor Coactivator NCOA3 Regulates UV Radiation–Induced DNA Damage and Melanoma Susceptibility

doi: 10.1158/0008-5472.can-20-3450

Figure Lengend Snippet: Figure 5. Effects of NCOA3 levels on UVR sensitivity in human melanoma cell lines. A, Representative images of immunofluorescence detection of NCOA3 and XPC in C8161.9 cells treated with vehicle (DMSO) or bufalin (quantitation provided in Supplementary Fig. S3F). B, Cell survival analysis of UVC-exposed C8161.9 cells (in triplicate). C, Cell survival analysis of UVC-exposed D04 cells (in triplicate). D, Cell survival analysis of UVC-exposed Ma-Mel-12 cells (in triplicate). E, Colony formation assay of UVC-exposed C8161.9 cells previously treated with vehicle (DMSO) or bufalin. F, Colony formation assay of UVC-exposed D04 cells previously treated with vehicle (DMSO) or bufalin. G, Colony formation assay of UVC-exposed Ma-Mel-12 cells previously treated with vehicle (DMSO) or bufalin. H, Effects of XPC cDNA overexpression on colony formation of UVC-exposed C8161.9 cells previously treated with bufalin (all assays performed in triplicate). , P < 0.05 denotes statistically significant differences compared with control. Scale bars, 20 mm.

Article Snippet: To overexpress pcDNA3, human NCOA3 cDNA or T960T NCOA3 polymorphism cDNA in NHEM Mel-F-Neo cells and human XPC cDNA (or a control plasmid expressing enhanced GFP, or EGFP) in C8161.9 cells, lipofectamine 2000–mediated transfections (9 mL) of (3 mg) plasmids expressing control pcDNA3 or EGFP, human XPC cDNA (Addgene#39204), human NCOA3 (Genecopoeia), or T960T were carried out following manufacturer’s instructions (Thermo Fisher Scientific).

Techniques: Quantitation Assay, Colony Assay, Over Expression, Control

Journal: Cancer Research

Article Title: Nuclear Receptor Coactivator NCOA3 Regulates UV Radiation–Induced DNA Damage and Melanoma Susceptibility

doi: 10.1158/0008-5472.can-20-3450

Figure Lengend Snippet: Figure 6. Role of differential NCOA3 levels in UV and melanoma susceptibility. A, Representative images of immunofluorescence detection of various proteins in NHEM cells transfected with control (pcDNA) or human NCOA3 cDNA plasmids (quantitation provided in Supplementary Fig. S8A). B, Representative images of immuno- fluorescence detection of NCOA3 and gH2AX in NHEM cells transfected with control (pcDNA) or human NCOA3 cDNA plasmids with UVC treatment (quantitation provided in Supplementary Fig. S8B). C, Prevalence of the T960T NCOA3 polymorphism in a control population (364 cases) versus a familial melanoma cohort (97 cases; left), and in the European (E; HapMap-CEU) versus Sub-Saharan African populations (S-S A; HapMap-YRI; right). D, Western blot analysis of NCOA3 and GAPDH in NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T), or human wild-type NCOA3 cDNA (hNCOA3) plasmids (densitometric values provided were normalized to GAPDH). E, UVC sensitivity (in triplicate) of NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T), or human wild-type NCOA3 cDNA (hNCOA3) plasmids by cell survival analysis. F, ELISA assay (in triplicate) of 6-4PP from UVC-exposed NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T) or human wild-type NCOA3 cDNA (hNCOA3) plasmids. G, ELISA assay (in triplicate) of CPD from UVB-exposed NHEM cells transfected with control (pcDNA), human T960T NCOA3 polymorphism cDNA (hT960T), or human wild-type NCOA3 cDNA (hNCOA3) plasmids. , P < 0.05 denotes statistically significant differences compared with control. Scale bars, 20 mm.

Article Snippet: To overexpress pcDNA3, human NCOA3 cDNA or T960T NCOA3 polymorphism cDNA in NHEM Mel-F-Neo cells and human XPC cDNA (or a control plasmid expressing enhanced GFP, or EGFP) in C8161.9 cells, lipofectamine 2000–mediated transfections (9 mL) of (3 mg) plasmids expressing control pcDNA3 or EGFP, human XPC cDNA (Addgene#39204), human NCOA3 (Genecopoeia), or T960T were carried out following manufacturer’s instructions (Thermo Fisher Scientific).

Techniques: Transfection, Control, Quantitation Assay, Western Blot, Enzyme-linked Immunosorbent Assay