ith oligo-primers according to the manufacturer’s protocol. The gene expression of adiponectin and its receptors AdipoR1 and AdipoR2, IL1b, IL6, IL8, IL10, heme oxygenase 1, MMP1, MMP3, transforming growth factor b1, keratinocyte growth factor, and involucrin was detected by real-time PCR using the iCycler iQ detection 
system, SYBR Green, and specific primers. Primer sequences, annealing temperatures and efficiencies are presented in Materials and Methods Isolation and culture of human oral epithelial cells Healthy gingival tissues were obtained from six donors, who underwent tooth extraction and/or dentoalveolar surgery. Written informed parental consent and approval of the Ethics Committee of the University of Bonn were obtained. The gingival specimens were washed twice with phosphate buffered saline supplemented with 1% antibiotic and antimycotic solution and subsequently digested with collagenase 2 solution at 37uC ELISA The concentrations of IL1b, IL8 and MMP1 in culture supernatants at 24 h and 72 h were analyzed by a commercially available enzyme-linked immunoassay kit according to the manufacturer’s instructions. The absorbance was measured with a microplate reader at 450 nm. The data were normalized by the cell number, which was measured with an automatic cell counter. Regulatory Effects of Adiponectin PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22179927 Gene Adiponectin AdipoR1 AdipoR2 b-actin HMOX1 IL1b IL6 IL8 IL10 MMP1 MMP3 Involucrin TGFb1 KGF sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense Primer sequences 59-GCCTCTTCAAGAAGGACAAGGCTATG -39 59-CAGTTGGTGTCATGGTAGAGAAG -39 59-ACTGGAGCTGGCCTTTATGCTGC -39 59-AGAGAAGGGTGTCATCAGTACAGC -39 59-CCATAGGGCAGATAGGCTGGTTGA -39 59-CAGTGCATCCTCTTCACTGCAGC -39 59-CATGGATGATGATATCGCCGCG-39 59-ACATGATCTGGGTCATCTTCTCG-39 59-CCAGGCAGAGAATGCTGAGTTCAT-39 59-CCGTACCAGAAGGCCAGGTCC-39 59-ATGGCAGAAGTACCTGAGCTCGC-39 59-TTAGGAAGACACAAATTGCATGGTG-39 59-ATGAACTCCTTCTCCACAAGC-39 59-CTACATTTGCCGAAGAGCCC-39 59-ATGACTTCCAAGCTGGCCGTGG-39 59-TGAATTCTCAGCCCTCTTCAAAAAC-39 59-TTAAGGGTTACCTGGGTTGC-39 59-GCCTTGCTCTTGTTTTCACA-39 59-ATGCACAGCTTTCCTCCACTGC-39 59-CACTGGGCCACTATTTCTCCGC-39 59-ATCGATGCAGCCATTTCTGATAAGG-39 59-TCAACAATTAAGCCAGCTGTTACT-39 59-CCCAGCAACACACACTGCCAGT-39 59-GCTCAGGCAGTCCCTTTACAGCA-39 59-GAGCCCTGGACACCAACTAT-39 59-GACCTTGCTGTACTGCGTGT-39 59-AGTTGGAATTGTGGCAATCA-39 59-CCGTTGTGTGTCCATTTAGC-39 Efficiency 1.93 2.04 1.97 1.84 1.97 1.83 2.12 2.02 1.94 2.05 2.06 1.98 1.94 1.84 Annealing temperature 69uC 69uC 69uC 69uC 69uC 68uC 68uC 68uC 65uC 69uC 69uC 69uC 69uC 65uC doi:10.1371/journal.pone.0030716.t001 MedChemExpress PF-04447943 proliferation assay The epithelial cell proliferation was determined by using the PromoKine XTT Cell Proliferation Kit. Following stimulation with LPS and/or adiponectin for 24 h and 48 h, cells were incubated with XTT reaction solution for 4 h. Finally, the absorbance was measured by using a microplate reader at 490 nm. In-vitro wound healing assay In order to study the wound fill rate in vitro, we used an established in-vitro wound healing model. Briefly, epithelial cells were seeded onto 35 mm culture dishes and grown until confluence. Then, defined cell-free areas were created by disrupting the monolayers with sterile instruments in a standardized manner. Subsequently, medium was changed and cells were stimulated, as described above. Pictures of the wounded area were taken on