The canonical hexa-acylated LPS of Escherichia coli JM 83-wild type strain was used as the reference [66]. Cell culture

HGFs were https://www.selleckchem.com/products/azd5153.html obtained from Sciencell research laboratories (Carlsbad, CA, USA) and cultured according to the manufacturer’s instructions [67, 68]. Continuous subcultures up to 10th passage contained homogeneous, slim and spindle-shaped cells growing in characteristic swirls. Third to fourth passages of HGFs without any signs of senescence were used for all experiments as described in our previous study [4]. Stimulation of HGFs by heterogeneous P. gingivalis LPS The cells suspended at 105 cell/ml were seeded on six-well-plates and grown until Selleck Rabusertib confluent at 37°C with 5% CO2 in a culture medium for fibroblasts consisting of basal medium with 2% fetal bovine serum, penicillin/streptomycin (0.01% w/v) and fibroblast growth supplement. Once the cells were over 90% confluent, fibroblast medium (FM) was replaced entirely with serum free and animal component free-medium (CX-6258 solubility dmso FM-acf) for the dose- and time-dependent experiments. In the dose-dependent assay, cells were stimulated with P. gingivalis LPS1435/1449, P. gingivalis LPS1690 or E. coli LPS in the media containing various doses of LPS (0.001 μg/ml −10 μg/ml). Subsequently, 1 μg of LPS was selected as the appropriate

dose for the following time-dependent experiments. Cells were incubated with P. gingivalis LPS or E. coli LPS at 1 μg/ml and harvested at 2, 12, 24 and 48 h. Cells without LPS treatment were designated as the controls. Culture supernatants

were collected and centrifuged to remove the cellular debris and stored at −70°C for Adenosine triphosphate subsequent protein assays. Cellular fraction was then washed with PBS and collected for mRNA and protein extraction. RNA extraction, cDNA synthesis and real-time qPCR Total RNA extraction, cDNA transcription and real-time qPCR for MMPs1-3 and TIMP-1 were performed as previously described [17]. In brief, total RNA was extracted from the homogenized HGFs using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions [35]. cDNA was synthesized by reverse transcriptase-PCR at 43°C for 90 min in a 20 μl of reaction mixture containing 1 μg of total RNA, 1 μl (200 U) of SuperScript™ First-Strand Synthesis System (Invitrogen Corp., Carlsbad, CA, USA), 0.5 μg of oligo dT-primer, first-strand buffer, 10 mM DTT, and 1 mM dNTPs. A control reaction was performed without reverse transcriptase for all samples to verify the absence of genomic DNA contamination. Real-time qPCR was then performed by using the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA) in at least three separate experiments.