This data was confirmed by western blotting and double immunofluorescence staining

h 1x diluted Giemsa reagent to identify cell colonies. The 3D matrigel colony formation assay was performed as previously described. The 96-well flat bottom plates were first coated with matrigel before being seeded with 200 transduced cells. Colonies were formed over a span of two weeks. 3 / 21 Role of p65 NF-B and Its Modulatory Mechanism in NPC Wound healing The wound healing assay was performed as described. The percentages of wound closure were calculated based on the formula: x 100. All wound healing experiments were conducted in triplicates and the average percentage wound closure was taken. Real-time cell migration assays Real-time cell migration monitoring was conducted using the xCELLigence system following the manufacturer’s MedChemExpress 2783-94-0 protocol. In brief, 8×104 cells were seeded into each well in the upper chamber of the CIM plate, which was coated with collagen. The lower chamber containing 10% serum media attracts the cells across the chambers, which will then be recorded real-time using a RTCA DP analyzer. Cell migration chamber assays The migration assay was performed as reported previously. In brief, 2 x 105 HONE1 cells in serum-free medium were seeded in the top chamber of a BD BioCoat Control 8.0m PET Membrane 24-well cell culture insert. The bottom chambers were filled with serum-enriched medium that acts as a chemoattractant. Cells were subsequently stained after 24 hours with 1% crystal violet. Cells that underwent migration were counted using an inverted light microscope and SPOT software 4.6. Human umbilical vein endothelial cell assay The tube formation assay was conducted as described using vector-alone and LTBP2 or FBLN2-conditioned media. HUVEC was first serum-starved for one hour prior to being seeded with 4 x 104 cells into each well of a flat bottom 96-well plate coated with 50l of matrigel. Conditioned media from the vector-alone or gene transfectants were then applied onto the cells and incubated for 68 hours. Quantitative reverse transcription-polymerase chain reaction Quantitative RT-PCR analysis was performed on a Step-One Plus PCR machine as described previously. The SYBR green PCR core reagent kit was used with gene-specific primers and GAPDH-specific primers as a control. Primers used are summarized in S3 Immunohistochemistry IHC staining for the CD34 marker on progenitor cells of blood vessels was performed using the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666110 standard streptavidin-biotin-peroxidase complex method as previously reported. Immunofluorescence staining Transduced HONE1 and HK1 cell lines were fixed in formalin for 10 min and permeabilized with 0.5% Triton X-100 in PBS as described. The phosphorylated p65 Serine 536 primary antibody was incubated with the cells overnight at 4C, followed by a FITC-conjugated 4 / 21 Role of p65 NF-B and Its Modulatory Mechanism in NPC goat anti-rabbit secondary antibody . Total IBa was labeled with Alexa Fluor 546 goat anti-mouse secondary antibody. Nuclei were stained with DAPI, cells were mounted onto glass slides with Slowfade Gold Antifade Reagent and viewed under a fluorescence inverted microscope or confocal microscope. NF-B p65 chemiluminescence transcription factor binding assay The NF-B/p65 transcription factor binding assay was carried out using the Millipore Universal EZ-TFA Chemiluminescent Transcription Factor Kit following the manufacturer’s protocol. In vivo tumorigenicity assay In vivo experiments were conducted as PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667157 reported previously. Cells were administered via subcutaneous injection into bo