Journal: Nucleic Acids Research
Article Title: Functional role of NF-IL6? and its sumoylation and acetylation modifications in promoter activation of cyclooxygenase 2 gene
Figure Lengend Snippet: NF-IL6β plays a role in transcription of cox-2 gene. ( A ) Overexpression of NF-IL6β enhances the cox-2 reporter activity. A431 cells were transfected with 0.2 µg of reporter vector pXC80 carrying cox-2 promoter (−80/+49 bp) together with expression vector of NF-IL6β in 1 ml of Opti-MEM medium. After medium change, cells were treated with or without EGF for 13 h. Cell lysates were then prepared, and luciferase activity was assayed. ( B ) Silencing of NF-IL6β expression attenuates EGF-induced cox-2 reporter activity. Cells were transfected with 1 µg of pXC918 carrying cox-2 promoter (−918/+49 bp) together with 1 µg of pSi-C or pSi-I. The upper panel shows that the transfection of NF-IL6β expression vectors attenuated the pXC918 reporter activity. The lower panel shows that the transcriptional products of cox-2 and NF-IL6β genes which were examined by RT–PCR analysis. The pSi-C represents the p Silencer ™ negative control plasmid. The pSi-I indicates the specific NF-IL6β knockdown expression vector. ( C ) Reduction of NF-IL6β expression decrease COX-2 expression. The transfection of pcDNA3/HA-NF-IL6β with pSi-C or pSi-I was performed in HeLa cells. Cellular lysates were harvested and analysed by western blotting probed with α-HA, α-COX-2 and α-β-actin antibodies. β-Actin was used as an internal loading control. ( D ) Silencing of c-Jun expression attenuates NF-IL6β-induced cox-2 reporter activity. A431 cells were transfected with 0.2 µg of pXC80, together with 0.5 µg of expression vector pSUPERc-Jun siRNA and 0.2 µg of each expression vector of c-Jun and NF-IL6β in 1 ml of Opti-MEM medium. Statistical significance between pSUPERc-Jun siRNA-transfected and untransfected cells was analysed by Student's t -test. ( E ) Cooperation of NF-IL6β with c-Jun in promoter activation of cox-2 gene. A431 cells were transfected with 0.2 µg of pXC80 reporter vector together with 0.05 µg of expression vector of NF/IL6β and 0.02 µg of expression vector of c-Jun in 1 ml of Opti-MEM medium. Cell lysates were then prepared, and luciferase activity was assayed. Statistical significance between c-Jun-transfected and untransfected cells was analysed by Student's t -test.
Article Snippet: NF-IL6β was generated from human liver cDNA library by PCR using BD Advantage GC™ PCR kit and using the following oligonucleotides: 5′-CGGGATCCAGCGCCGCGCTCTTCAGCCTG-3′ and 5′-GGCCTCGAGGCCGCGCGTTACCGGCAGTC-3′.
Techniques: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Activation Assay