Mmp 7

Quantitative PCR (qPCR) was used to profile MMP expression in cancer cells and whether or not BAs had an effect on MMP gene expression. Briefly, total cellular RNA was isolated from cancer cells using RiboPure kit from Ambion (Austin, TX, USA) according to manufacturer’s protocol. The RNA was quantified on the Nano Drop 8000 (Thermo Scientific, Wilmington, DE, USA) where the quality of mRNA was evaluated by the OD260/OD280 ratio. Only samples with a ratio value of ~2.0 were used. For reverse transcription reaction, 1 µg of total RNA (High Capacity cDNA Archive Kit) was used and 10 ng of transcribed DNA was spent for each quantitative PCR reaction. As target probes, TaqMan MGB human MMP-1, MMP-2, MMP-7, MMP-9 and MMP-10 (Applied Biosystems, Dublin, Ireland) were used along with a housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold inductions were calculated using the comparative CT method as described in the ABI Prism manual using GAPDH as the internal control [41 (link)]. Gene expression levels of the MMPs were expressed as the cycle threshold (C_t) value (MMP)/C_t value (GAPDH). GAPDH levels (C_t values) were similar across all cell lines. Values are presented as fold change in gene expression relative to the control group, which was normalized to 1.

Total RNA was extracted from peripheral blood mononuclear cells (PBMC), primary monocytes and atherosclerotic plaques and non-atherosclerotic vessels using RNeasy spin columns (QIAGEN, Hilden, Germany), subjected to DNase I treatment, and stored at −80°C until further analysis. cDNA was synthesized using high-capacity cDNA archive kits (Applied Biosystems, Foster City, CA). Gene expression was assessed using TaqMan assays; MMP-7: Assay ID Hs01042796_m1, CD45: AssayID Hs00365634_g1 and CD68 AssayID: Hs00154355_m1 (Applied Biosystems) on the ABI Prism 7500 instrument (Applied Biosystems). Gene expression of the reference gene β-actin was used for normalization.

SAS cells were seeded in 6 well plates and treated with NDV at an MOI of 0.1 for 48 hr. The media was then removed and the whole cell lysate was collected with RIPA lysis buffer containing 1x ProteoGuard EDTA free protease inhibitor cocktail (Clontech, USA). Cell lysates were collected by centrifugation at maximum rpm for 15 minutes at 4 °C and the supernatant was stored at −80 °C till use. Caspase 3, poly ADP ribose polymerase (PARP), cytochrome C, anti-GFP, β-catenin, cyclin D1, c-Myc, MMP-7, p-Akt (Ser473), β-actin (Thermo Scientific, USA), total GSK-3β, p-GSK-3β (Ser9) (Cell Signaling Technology, USA) and histone (H3) (BioBharati Life Sciences, India) antibodies were used to develop the immunoblots. The polyclonal antiserum generated against NDV in SPF chickens was used to detect the viral specific proteins. The β-actin was used as an internal control for all the immunoblots.