Journal: PLoS ONE
Article Title: Hepatitis C Virus Induced Endothelial Inflammatory Response Depends on the Functional Expression of TNFα Receptor Subtype 2
Figure Lengend Snippet: Amplification of poly (I:C)-mediated induction of cytokines, chemokines and adhesion molecules by TNFα. HMEC were grown in culture medium for 24 hours and additionally incubated in medium (basal) for different time intervals (6, 12, 24 hours) or grown in culture medium for 24 hours and additionally incubated with TNFα (TNFα) for different time intervals (6, 12, 24 hours) or incubated with poly (I:C) for 24 hours and then grown in medium (poly (I:C)) or stimulated with TNFα (poly (I:C) + TNFα) for different time intervals (6, 12, 24 hours). mRNA expression of selected cytokines and chemokines IL-6 (A), IL-8 (C), IP-10 (E), RANTES (G), MCP-1 (H), IFN-β (I), MCSF (J), ICAM-1 (K) and VCAM-1 (L) was analyzed by RT-PCR. Protein synthesis of selected targets IL-6 (B), IL-8 (D) and IP-10 (F) was also confirmed by ELISA. Results are given as means ± SD of three experiments done in parallel for each condition and rRNA served as the reference gene. Comparable results were obtained in two series of independent experiments. Statistically significant differences to the control are depicted with * = p≤0.05, ** = p≤0.01.
Article Snippet: Sequences were used as indicated or pre-developed Taq Man assay reagents or primers and probes were purchased from Applied Biosystems: NM_003265.2, HS00152933_m1 (human TLR3), NM_014314.3, Hs00204833_m1 (human RIG-I), NM_022168.2, Hs0170332_m1 (human MDA5), NM_000600.3, Hs00174131_m1 (human IL-6), NM_000584.2, Hs00174103_m1 (human IL-8), NM_002985.2, Hs00174575_m1 (human RANTES), NM_002982.3, Hs00234140_m1, (human MCP-1), NM_001565.2, Hs00171042_m1 (human IP-10), FP: CCT TCC TCC TGT CTG ATG GA ; RP: ACT GGT TGC CAT CAA ACT CC ; T1: 6FAM CAG ACA TGA CTT TGG ATT TCC CCA GG (human IFN-α), NM_002176.2, Hs00277188_s1 (human IFN-β), NM_172210.2, NM_172211.2, NM_172212.2, NM_000757.4 (human MCSF), NM_000201.2, Hs00164932_m1, (human ICAM-1), NM_001078.3, NM_001199834.1, NM_080682.2, Hs00365486_m1 (human VCAM-1), NM_000594.2, Hs00174128_m1 (human TNFα), NM_001065.3, Hs00533560_m1 (human TNFR1), NM_001066.2, Hs00153550_m1 (human TNFR2) and M33197 (human GAPDH).
Techniques: Amplification, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay