The following primers were used in PCR: and for P2X1; and for P2X2; and for P2X3; and for P2X4; and for P2X5; and for P2X6; and for P2X7; and and for pannexin1. The PCR cycling protocol for all those primers was 94C at 5 s, 55C at 5 s and 68C at 15 s. P2X4, P2X7, or pannexin-1 complex blocks IL-1 secretion in ATCC 33277 was cultured anaerobically for 24 h at 37C in trypticase soy broth (TSB) supplemented with yeast extract (1 mg/ml), hemin (5 g/ml) and menadione (1 g/ml) and utilized for contamination as explained . The human immortalized gingival keratinocyte (HIGK) cell collection , was obtained as previously explained , . Cells were E 2012 cultured in serum-free defined keratinocyte-SFM (Gibco) at 37C in a humidified incubator made up of 5% CO2. Main GEC were obtained after oral surgery from healthy gingival tissue as previously explained . Cells were cultured as E 2012 monolayers in serum-free keratinocyte growth medium (KGM) (Lonza) at 37C in 5% CO2. Main GEC were utilized for experimentation at 75C80% confluence and cultured for 24 h or 48 h before contamination with at a multiplicity of contamination (M.O.I.) of 100 . ATP, ADP, UTP, oxATP, PPADS, and probenecid were from Sigma-Aldrich. AMP was from Santa Cruz Biotech. 5-BDBD was from Tocris Bioscience. All primers were E 2012 purchased from Fisher Scientific. Antibodies against P2X4 (APR-002) and P2X7 (APR-008) were obtained from Alomone Labs. RNA Extraction, Reverse Transcription PCR, and Quantitative PCR Total RNA was isolated from 106 HIGK cells using RNeasy Mini kit (Qiagen) according to the manufacturers protocol. cDNA was amplified from 2 g RNA by random hexamers E 2012 using TagMan Reverse Transcription Reagents kit (Applied Biosystems). The following primers were used in PCR: and for P2X1; and for P2X2; and for P2X3; and for P2X4; and for P2X5; and for P2X6; and for P2X7; and and for pannexin1. The PCR cycling protocol for all those primers was 94C at 5 s, 55C at 5 s and 68C at 15 s. The protocol was repeated for 40 cycles and included an initial 5 min enzyme activation step at 94C and a final 10 min extension step at 72C. PCR products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. Quantitative PCR (qPCR) was carried out with 1/50 of the cDNA preparation in the Mx3000P (Stratagene) in 25 l final volumes with the Amazing QPCR Master Mix (Stratagene). cDNA was amplified using 200 nM of each specific sense and antisense primers. Quantitative PCR was conducted at 95C for 10 min, followed by 40 cycles at 95C for 30 s, 55C for 1 min and 72C for 30 s. The expression levels of P2X4, P2X7, and pannexin-1 were normalized to GAPDH by the comparative cycle threshold method, as described by the manufacturer (Stratagene). The primers for the genes coding P2X4, P2X7, and pannexin-1 were as above. For GAPDH, the primers were: and prospects to expression of pro-IL-1 and its accumulation within the infected cell. However, secretion of IL-1 requires BMP6 a second transmission, such as the danger transmission ATP, in order to activate the NLRP3 inflammasome and caspase-1, allowing processing and secretion of the mature IL-1 . Given the unexpected observation that P2X4 can modulate ATP-dependent caspase-1 activation in the immortalized HIGK cells, we examined whether a similar effect could be observed in immortalized (HIGK) cells and main GEC during contamination with contamination alone nor contamination combined with 100 M ATP treatment could induce IL-1 secretion by HIGK cells. Only infected cells treated with 3 mM ATP, but not other nucleotides, could promote Il-1 secretion (Physique 6A). Similarly, using main GEC, we found that ATP, but not other nucleotides, could promote IL-1 secretion by infected cells (Physique 6C). We also consistently observed that main GEC produce and secrete higher levels of IL-1 than HIGK cells (Physique 6). Open in a separate window Physique 6 Abrogation of ATP-induced IL-1 secretion in ((contamination followed by 3 mM ATP treatment caused IL-1 secretion.