ADAMTS-12 and Trophoblast Invasion Figure 2. Regulatory effects of TGF-b1 and IL-1b on ADAMTS-12 mRNA expression levels in EVTs. (A) QC-PCR analysis of ADAMTS-12 mRNA levels in EVTs cultured in the presence of (i) TGF-b1 for 0–48 h, (ii) 0–10 ng/ml of TGF-b1 for 24 h, (iii) in the presence of vehicle, TGF-b1 with or without an antibody against TGF-b1 for 24 h. (B) Similar analyses following (i) 0–48 IL-1b, (ii) 0–1000 IU/ml of IL-1b for 24 h, (iii) in the presence of vehicle, IL-1b with or without an antibody directed against IL-1b for 24 h. Representative photomicrographs of the resultant ethidium bromide- stained gels are presented (t and c denote the target and competitive PCR transcripts respectively). The data are presented mean 6 SEM (n = 3; a = P#0.05 vs. untreated control; b = P#0.05 vs. cytokine alone). doi:10.1371/journal.pone.0018473.g002 the major protein species detected, exogenous expression of ADAMTS-12 also revealed a minor protein species of approxi- mately 110 kDa and 50 kDa. Utilizing our transwell invasion system, we next determined that the invasive capacity of JEG-3 cells exogenously expressing ADAMTS-12 was significantly and consis- tently higher than the control cultures (Figure 4A). Proteinase-dead ADAMTS-12 increases invasion of JEG-3 cells As a first step in characterizing the molecular mechanisms underlying ADAMTS-12-mediated cellular invasion, JEG-3 cells were stably transfected with the mammalian expression vector containing the proteinase-dead form of ADAMTS-12 (A12Mut) [21]. Similar to our preceding findings, a major ADAMTS-12 protein species (83 kDa) was readily detectable in these JEG-3 cells following transfection with this vector (Figure 4A). Furthermore, Matrigel invasion analysis demonstrated that there was no significant difference in the invasive capacity of these cells and those expressing A12FL(3.560.9 (A12Mut) vs. 5.4361.3 (A12FL); p#0.18), with both cell lines being consistently and significantly more invasive than the control cultures of JEG-3 cells (Figure 4A). ADAMTS-1 cannot mimic the effects of ADAMTS-12 induced invasion on JEG-3 cells To determine whether an increase in the expression levels of another ADAMTS subtype, other than ADAMTS-12, could PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e18473