Cell proliferation assays for MC3T3-E1 cells were performed for 2 weeks. In brief, 104 cells were seeded on 12-well plates. The cells were harvested at indicated time points by trypsin/EDTA and then resuspended in 1 ml culture medium. After centrifuga- tion, cell pellets were resuspended in 1% FBS containing 70% ethanol and kept at 4uC until analysis. Prior to use, the cell suspensions were exchanged into 1 ml fetal bovine serum (PBS) and the cell number was counted with a flow cytometer (FACSCalibur, BD bioscience, NJ, USA). Differentiation of MC3T3-E1 osteoblast cells was carried out by incubation in differentiation medium (50 mg/ml ascorbic acid, 10 mM beta-glycerophosphate, and 1028 M dexamethasone in alpha-MEM). The medium was changed every 2–3 days. Cell differentiation was identified by ALP staining. In general, differentiated cell layers were washed with PBS and fixed with neutral formalin (10% formaldehyde in PBS) for 15 min. After removal of fixation solution and successive incubation in distilled water for 15 min, the cell layers were stained with a staining solution (1 mg/ml Naphthol AS-MX-PO4; 6 mg/ml fast red violet LB salt, in 10 mM Tris-HCl, pH 8.0) at 37uC for 45 min. After removal of staining solution and rinsing 3 times with water, the stained cells were kept in distilled water for microscopic examination. Construct generation The nephronectin cDNA was a kind gift of Dr. Louis. F. Reichardt. The primer sets used to amplify the two constructs of nephronectin are as follows. Npnt-N (59cccgggctcgagtggcccagg- caaatagtttct) and Npnt-C (59cccgggtctagatcagcagcgacctcttttcaa) were used to clone nephronectin coding sequence. Npnt-N and Npnt-end (59cccgggtctagaaggaaattccatgcccaccct) were used to clone nephronectin coding sequence plus the 39UTR. MAM-N (59cccgggctcgaggtgtatatccccaaagtc) and Npnt-C were used to cloned the fragment containing the MAM domain. A link protein leading peptide (LP) was attached to the N-terminus of the construct to allow product secretion and detection by the monoclonal antibody 4B6 [61,62]. The sequences of the generated constructs were confirmed by restriction digestion and sequencing. A luciferase reporter vector (pMir-Report; Ambion) was used to generate luciferase constructs containing either GalNT7 39UTR or mutant GalNT7 39UTR. The 39UTR of GalNT7 was cloned using two primers, GalNT7-SacI (59cccggggagctcgcccttgttagat- taggccttata) and GalNT7-MluI (59gggcccacgcgtcatttgcaatattt ggttttcctaa), by PCR. The PCR products were then digested with SacI and MluI and the fragment was inserted into a SacI- and MluI-digested pMir-Report Luciferase plasmid (Ambion), to obtain a luciferase construct, luc-Gal-UTR. The mutant construct was generated with two primers, GalNT7-SacI and GalNT7- MluI-mut (59gggcccacgcgtcatttgcaatatttggtttaggaaatagg) using the same method. —–– To generate a small interfering RNA against nephronectin, two primers (59ccaggtagatctaggactgaactgtgtgtatattcaagagtatacacacagtt- cagtccgactgccgaccagcagagcag and 59gggctaaagcttaaaaaacagtac- cactacacgagtagagaacattactcgtgtagtggtactgctgctctgctggtcggcagtc) were designed for PCR. The PCR product was digested with BglII and HindIII and inserted into the BglII- and HindIII-digested BluGFP vector [53], resulting in a construct expressing siRNA against nephronectin. The construct for miR-378 was designed by us and synthesized by Top Gene Technologies (Montreal, Canada). In brief, the pre- miRNA of miR-378 was ligated into the mammalian expression vector BluGFP that contains a Bluescript backbone, a CMV promoter driving the GFP, and a promoter driving the expression of miR-378 as previous described [53,63]. miR-378 in Differentiation Western blot and proteomic analysis For western blot analysis, cells were harvested by 10 mM EDTA at 60–80 % confluence and washed three times with PBS using centrifugation. The cells (16106 cells) were disrupted and kept on ice for 15 min. The lysate was then centrifuged at maximum speed using a desktop centrifuge at 4 degrees for 10 min. Protein concentrations were quantified by a protein assay kit (Bio-Rad Laboratories Ltd., ON, Canada). Protein lysate (100 mg) was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and the separated proteins were transferred onto a nitrocellulose membrane followed by blocking and incubation in a primary monoclonal antibody overnight at 4 degrees. After washing, the membrane was incubated with HRP-conjugated goat-anti-mouse secondary antibody for 1 hour at room temper- ature followed by ECL detection. For proteomic analysis, the cells were cultured for three days under normal growth conditions. The cells were detached from the culture dishes by 1 mM EDTA and then washed three times with PBS. Cell pellets were then delivered on dry ice for proteomic analysis by WEMB Biochem Inc (Richmond Hill, ON, Canada). Characterization of protein glycosylation To characterize the type of post-translational modification of nephronectin, the conditioned medium containing fully glycosy- lated nephronectin was digested by different de-glycosylation enzymes. The size of the resulting protein was then observed by western blot. The conditioned medium (30 ul) was treated with 10 ml enzyme solution containing 2 units PNGase F (to release N- glycan), 2.5 units O-glycosidase (to release O-glycan), 0.01 units sialidase (to release terminal sialic acid) or 0.1 units keratinase (to release keratin sulfate) at 37uC overnight. To completely remove the O-glycan from the protein core, the combined digestion by O- glycosidase and sialidase was achieved by the same incubation conditions. Statistical Analysis The results (mean values6SD) of all experiments were subjected to statistical analysis by the t-test. The level of significance was set at P,0.05. Supporting Information Figure S1 Effects of nephronectin on MC3T3-E1 cell activities. (A) Time course analysis showing the different effects of Npnt and Npnt+39 transfections on MC3T3-E1 differentiation: early stages, Npnt.Npnt+39; late stages, Npnt,Npnt+39. (B) The cell lines were cultured under nodule-inducing conditions over the three different phases of osteoblast development. Bone nodules formed on day 56 are shown after silver nitrate staining for the detection of bone nodules. The existence of miR-378 slightly enhanced cell proliferation in Npnt-transfected cells (C), but inhibited cell proliferation in Npnt+39-transfected cells (D). Found at: doi:10.1371/journal.pone.0007535.s001 (1.52 MB PPT) Figure S2 Targeting of GalNT7 by miR-378. (A) A fragment of GalNT7 39UTR was inserted into the luciferase report vector pMir-Report producing a construct named luc-Gal-UTR. The potential miR-378 target sequence was labelled in blue. Mutations labelled in red color were generated in the miR-378 target sequence producing a mutant construct named luc-Gal-mut. (B) MT-1 cells stably transfected with GFP and miR-378 were immunostained with anti-GalNT7 antibody, followed by confocal microscopic analysis. While both types of cells expressed GFP (green), miR-378-transfected cells expressed lower levels of PLoS ONE | www.plosone.org 12 October 2009 | Volume 4 | Issue 10 | e7535