Recombinant human ctgf

The transdifferentiation assay was performed as described before.^{41 (link)} Briefly, ATDC5 cells were cultured in chondrogenic differentiation medium for 7 days and thereafter cultured in osteogenic differentiation medium containing 50 ng/mL recombinant human Ctgf (PeproTech Inc., Rocky Hill, NJ, USA). Cells were harvested after 24 and 48 h of osteogenic differentiation, respectively. RNA from cell lysates was isolated using the RNeasy Mini Kit and cDNA was generated using Omniscript Reverse Transcriptase (both Qiagen). qRT-PCR expression analysis was performed using Platinum™ SYBR™ Green qPCR SuperMix-UDG and ROX™ Reference Dye (both Thermo Fisher Scientific). The primer sequences of the analyzed genes are shown in Table S1. B2m expression was used as the reference housekeeping gene. Data analysis was performed according to the delta-delta comparative threshold cycle (2^-ΔΔCT) method, and results are shown as fold-change expression values relative to controls.

For dermal fibroblastic differentiation, when the cells reached 80%-90% confluence, the culture medium was changed to the differentiation medium supplemented with 100 ng/mL recombinant human CTGF (Peprotech, Rocky Hill, NJ, USA) and 50 µg/mL ascorbic acid (Sigma, Minato City, MI, USA). The differentiation medium was changed every 2 days for 7 and 14 days.

Anti-rabbit and anti-mouse IgG-conjugated horseradish peroxidase, rabbit polyclonal antibody specific for α-tubulin, and mouse monoclonal antibodies specific for CTGF, MMP-2, and MMP-3 were purchased from Biotechnology (Santa Cruz, CA). ON-TARGETplus siRNA of MMP2, MMP3, and control were purchased from Dharmacon Research (Lafayette, CO). PD98059, U0126 and human MMP-2 and MMP-3 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Calbiochem (San Diego, CA). Recombinant human CTGF was purchased from PeproTech (Rocky Hill, NJ). MiRNome microRNA Profilers QuantiMir™ kit was from System Biosciences (SBI) (Mountain View, CA). MiR-519d mimic and inhibitor were purchased from Invitrogen (Carlsbad, CA). MEK1 dominant-negative mutant was a gift from Dr. W. M. Fu (National Taiwan University at Taipei). ERK2 dominant-negative mutant was a gift from Dr. M. Cobb (Southwestern Medical Center, Dallas, TX), all other chemicals purchased from Sigma-Aldrich (St. Louis, MO).