Animal Protocol and Nucleic Acid Isolation All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Allegheny General Hospital, Pittsburgh, PA and followed guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The tissue harvesting protocol for our New Zealand white rabbits, RNA isolation/ storage from fetal and adult tissues was performed as previously described [11,12]. The quality and quantity of total RNA extracted from tissues and fibroblasts were determined by measuring the OD 260/OD 280 ratio using the ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE) and by capillary electrophoresis with the Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Palo Alto, CA). Dermal Fibroblast Culture New Zealand white rabbits were anesthetized and freshly excised skin specimens (around 1 sq cm) from adult and fetal rabbits (gestational age 21 days) were minced into small pieces within 30 minutes after dissection. These tissue pieces were washed exclusively in PBS containing 1% antibiotic/antimycotic solution (Sigma, St Louis, MO) and then placed in the RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution. The cultures were left undisturbed for a week at 37C with 5% CO2 supplement. The outgrowing fibroblasts seen after a week from these primary cultures were sub- cultured immediately using 0.5% Trypsin-EDTA (Invitrogen Corp.) Construction of siRNAs and Transfections Short interfering RNA (siRNA) duplexes were synthesized and purified by Integrated DNA Technologies (Coralville, IA). The siRNA sequences targeting rabbit CCT-eta, CCT-beta and alpha-SMA are shown in Table 1. A ‘‘scrambled’’ siRNA with an identical base pair composition but randomized sequence was also obtained as control for both the CCT-eta and CCT-beta subunits and is also shown in Table 1. A non-specific control siRNA from Abcam Inc. (Cambridge, MA) was used during a- SMA siRNA transfection. These siRNAs were employed in adult fibroblasts in transient transfection experiments wherein a) fibroblast mobility was assayed in an in vitro wounding protocol, and b) cellular contractility was assayed through traction force microscopy. Table 1. List of siRNA sequences used to target the genes of interest. Gene Rabbit CCT-eta Rabbit CCT-beta Rabbit a-SMA Sequences Sense-59 rGrArArCrGrArUrUrCrArGrUrArGrUrGrGrCrUTT 39 Antisense-59 rArGrCrCrArCrUrArCrUrGrArArUrCrGrUrUrCTT 39 Sense-59rGrGrArGrArArArGrUrUrGrArArCrGrUrArUrUTT-39 Antisense-59rArArUrArCrGrUrUrCrArArCrUrUrUrCrUrCrCTT39 Sense- 59rArGrArGrArArArUrUrGrUrGrCrUrArUrGrUrCTT39 Antisense-59rGrArCrArUrArGrCrArCrArArUrUrUrCrUrCrUTT39 Scramble Control Sense-59rGrArArcrGrArUrUrCrGrArArUrGrCrUrGrGrUTT39 CCT-eta Antisense-59rArCrCrArGrCrArUrUrCrGrArArUrCrGrUrUrCTT39 Scramble Control Sense-59rUrGrArArGrArArGrGrArArGrUrUrGrCrUrArUTT39 CCT-beta Antisense-59rArUrArGrCrArArCrUrUrCrCrUrUrCrUrUrCrATT39 doi:10.1371/journal.pone.0010063.t001 CCT-eta & Fibroblast Phenotype Rabbit adult fibroblasts were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. Transfection of siRNAs was performed with the manufacturer’s protocol using Lipofectamine 2000. Briefly, 7.5 ml of 20 mM siRNA was mixed with 200 ml of Opti-MEM; 4 ml of Lipofectamine 2000 was diluted into 200 ml of Opti-MEM and incubated at room temperature for 5 min. After the incubation, the diluted Lipofectamine 2000 was combined with the diluted siRNAs and then incubated for an additional 20 min (siRNA sequences targeting both CCT subunits and a-SMA were used at a concentration of 150 pM). A total of 400 ml of siRNA-Lipofectamine 2000 complexes was added to each well of cultured rabbit adult fibroblasts at <90% confluence in a six well plate. After 24 h incubation at 37C the cells were switched to quiescent media (RPMI 1640 medium containing 0.1% dialyzed FBS along with antibiotics) and left for 48 h. After 48 h of incubation in quiescent media cells were subjected to the in vitro wounding protocol described below; at this same time, cell populations were also stimulated either with EGF (1 nM)/ PDGF (200 nM) or control. Thus, during the period of cell motility assayed these growth factors (or control, that is, no treatment) were continuously present. To validate the sequence-specificity of gene knock-down in vitro, after transfection the fibroblasts were analyzed for protein and mRNA downregulation by immunoblots and real- time RT-PCR. Quantitative Real Time RT-PCR Total RNA isolated (RNeasy Micro Kit, Qiagen Inc., Valencia, CA) from rabbit adult fibroblasts after CCT-eta , CCT-beta and a-SMA siRNA transfection was subjected to real time RT-PCR to determine the abundance of these two CCT subunit messages. Primers and probes for these assays were designed by Primer Express software (Applied Biosystems, Foster City, CA) as described previously (Satish et al., 2010 manuscript submitted). The primers were obtained from Integrated DNA Technologies and the Taqman probes were purchased from Applied Biosystems. The experimental conditions for RT-PCR were carried out exactly as described previously [14]. Using the comparative critical cycle (Ct) method and using GAPDH as the endogenous control, the expression levels of the target gene products were normalized and the relative abundance was calculated. Data were analyzed using the 7900 HT SDS software version 2.1 provided by Applied Biosystems. Total RNA extracted from rabbit adult and fetal fibroblasts was similarly subjected to these quantitative comparative RT-PCR assays to determine the relative mRNA expression levels of CCT- eta, CCT-beta and a-SMA in these cell types at baseline. Immunoblotting Rabbit adult fibroblasts were lysed using SDS-sample buffer and proteins were analyzed after siRNA transfection against CCT-eta and CCT-beta. Proteins from equal volumes of cytosol from the various experimental conditions were resolved by SDS-PAGE and analyzed by immunoblotting with antibodies specific for CCT- eta, CCT-beta, and a-SMA. Antibody against GAPDH was used as a loading control. Equal amounts of protein lysates isolated from non-transfected control rabbit adult and fetal fibroblasts were also subjected to SDS-PAGE and probed with antibodies specific for CCT-eta, CCT-beta and a-SMA with GAPDH serving as a loading control. Cell Migration Assay Rabbit adult fibroblasts were plated on 6-well plastic dishes and grown to confluence in RPMI 1640 supplemented with 10% FBS. The cells were quiesced for 48 h in the media containing 0.1% PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10063