. The Phototope HRP Western Blot Detection System, including anti mouse IgGs, HRP linked antibodies, a biotinylated protein lad der, 20�� LumiGLO Reagent and 20�� pero ide, was pur chased from Cell Signaling Technology. The Anne in V FITC Propidium Iodide Flow Cytometry Assay Kit was purchased from Invitrogen. Antibodies directed against gC1qR, phosphorylated p38 MAPK, phosphorylated JNK, total p38 MAPK, total JNK and actin were purchased from Santa Cruz and Cell Signaling Technology. pcDNA HPV 16 E2 and pcDNA HPV 16 E2 mutant plasmids were kindly supplied by Hangzhou Hibio Bio tech Co, Ltd. gC1qR small interfering RNA and negative siRNA were synthesised by Wuhan Genesil Biotechnology Co, Ltd. Cell culture supplies were purchased from Life Technologies.

Unless otherwise specified, all of the other reagents were of analytical grade. C33a And SiHa cell culture and DNA Dacomitinib transfection conditions C33a and SiHa cells were grown in Dulbeccos modified Eagle medium, supplemented with 10% foetal bovine serum, 1% nonessen tial amino acids, and 2 mM glutamine. The cells were maintained in the presence of 5% CO2 at 37 C. Comple mentary DNA encoding HPV 16 E2 was cloned in frame using BamHI EcoRI sites into the pcDNA 3. 1 e pression plasmid. The resulting pcDNA HPV 16 E2 vector was then transfected into C33a and SiHa cells. Twenty four hours after plating, the cells were serum starved for an additional 24 h to quiescence. Following serum starvation, the cells were transfected using Lipofectamine reagent according to the vendors protocol. Briefly, 0. 05 1.

5 ug ml plasmid DNA and 12 ug ml Lipofectamine were diluted in serum free DMEM. After incubation for 30 min at 37 C, DNA liposome comple es were added dropwise to each culture dish and incubated at 37 C in a 5% CO2 atmos phere for 12 h. Following transfection, the cells were cul tured in serum free DMEM. Reporter gene levels were normalised to total protein, and each e periment was inde pendently performed three to five times. gC1qR SiRNA e pressing plasmid construction We designed siRNA to target the 408 426 nucleotide portion of human gC1qR mRNA. A gC1qR siRNA e pressing plasmid was constructed using pGenesil 1 as the vector backbone. BamHI and HindIII restriction site overhangs were located near the 5 end of the two oligonucleotides. a 6 nucleotide poly T tract recognised as an RNA pol III termination signal was lo cated at the 3 end of the siRNA template.

The siRNA was synthesised, annealed and ligated into the BamHI and HindIII restriction sites in the pGenesil 1 e pression vector. A vector containing siRNA for an unrelated gene was used as a negative control. Real time quantitative polymerase chain reaction Total RNA was isolated from tissue using Trizol rea gent according to the manufacturers instructions. Isolated RNA was then DNase treated and reverse transcribed according to the manufacturers instructions. Quantitative real time PCR was performed using an ABI PRISM 7300 sequence detec tion system with t