Adam17

Total RNA was isolated from cultured cells using the Total RNA Isolation NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany). cDNA was reverse transcribed from 1 μg RNA (QuantiTect Reverse Transcription kit; Qiagen, Hilden, Germany). 2 μl of the cDNA (diluted 1:15) were used in PCR reactions consisting of 5 μl 2x QuantiTect SYBR Green buffer (Qiagen) and 3 μl primer mix. Primers used were VE-cadherin (Hs_CDH5_5_SG; #QT00013244), ADAM10 (Hs_ADAM10_1_SG; #QT00032641), ADAM17 (Hs_ADAM17_1_SG; #QT00055580), and GAPDH (Hs_GAPDH_2_SG; #QT01192646) (all from Qiagen). Samples were run in triplicates on a 7900HT real-time PCR system (Applied Biosystems, Darmstadt, Germany). Data were analyzed using the SDS software (Applied Biosystems). In each sample, expression levels were normalized using the mRNA expression of the housekeeping gene GAPDH.

Gene expressions of il-6 and membrane-bound proteases adam10 and adam17 (both purchased from Qiagen) in hPDL fibroblasts as well as expressions of opg and rankl in phAOBs were analyzed by real-time polymerase chain reaction (rtPCR). RNA was isolated using the commercially available RNeasy Protect Mini Kit (Qiagen) according to the manufacturer's instructions. RNA concentration was measured using a NanoDrop^ⓇND-1000 spectrophotometer at 260 nm wavelength (NanoDrop Technologies, Wilmington, DE, USA) and consecutively transcribed to cDNA using the iScript^TM Select cDNA Synthesis Kit (Bio-Rad Laboratories, Munich, Germany). Afterwards, 1 μl of cDNA, 2.5 μl of the specific primer (Metabion, Martinsried, Germany or Qiagen according to Table 1), 12.5 μl of QuantiTect SYBR Green Master Mix (Qiagen) and 9 μl of nuclease free water were mixed and gene expression was detected in the iCycler iQ^TM real-time PCR detection system (Bio-Rad Laboratories). Protocol for rtPCR was as follows: an initial denaturation step at 95°C for 5 min was followed by 40 cycles of 10 s at 95°C, 30 s at annealing temperatures specific for the primers, and 30 s at 72°C for elongation. Glycerinaldehyd-3-phosphate dehydrogenase (gapdh) in phAOBs and ribosomal protein L22 (rpl22) in hPDL fibroblasts served as housekeeping genes for data normalization.