Oligo dt

Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA) according the manufacturer’s instructions. Briefly, 1 μg of total RNA was incubated with 0.5 μg of oligo dT (MD.Bio., Taipei, Taiwan) at 70 °C for 15 min. Then, the RNA was mixed with buffer containing 0.25 mM dNTPs (MD. Bio., Taipei, Taiwan), 20 U of RNasin I Plus RNase Inhibitor (Promega, WI, USA) and 20 U of M-MLV Reverse Transcriptase (Promega) and incubated at 42 °C for 90 min for cDNA synthesis. This mixture was then used for specific cDNA amplification in a GeneAmp PCR system 2400 (Perkin Elmer, Waltham, MA, USA).
For mRNA quantification, Real-time PCR was performed using a standard LightCycler 480 SYBR Green I Master protocol on an LightCycler 96 System Roche, Basel, Switzerland). The 10 μl PCR included 2 μl RT product, 5 μl 2 × SyberGreen PCR Mix, 0.5 μl 10 μM forward primer, 0.5 μl 10 μM reverse primer and 2 μl ddH₂O. All reactions were run in triplicate. The cycle number at which the reaction crossed the threshold cycle (Ct) was determined for each gene and the relative amount of each gene to GAPDH was described using the equation 2^ΔCt where ΔCt=(Ct_{interested gene} – Ct_GAPDH).

Recombinant human visfatin was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). VEGF-C (sc-9047) and IgG (sc-69786) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The cell culture mediums Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 were purchased from Gibco Life Technologies Corporation (Grand Island, NY, USA). Chloroform and isopropanol were purchased from J.T. Baker (NJ, USA). Oligo-dT was obtained from MDBio Inc. (Gaithersburg, Maryland, USA). Dithiothreitol (DTT), dNTP, MMLV and 5X first-strand buffer were purchased from Invitrogen Corporation (Carlsbad, California, USA). Taqman^® One-Step PCR Master Mix, qPCR primers and probes were bought from Applied Biosystems (Foster City, CA, USA). A BCA protein assay kit was obtained from Pierce (Meridian Rd. Rockford, IL 61101 USA). Tri’s buffer, 30% acrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Amresco Inc (6681 Cochran Rd, Solon, Ohio, USA). The detailed source of inhibitors and siRNAs are listed in Supplementary Tables 1 and 2. All other chemical reagents not already mentioned were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer's instructions. Briefly, 1 μg of total RNA was incubated with 0.5 μg of oligo dT (MD Bio., Taipei, Taiwan) at 70 °C for 15 min. Then, the RNA was mixed with buffer containing 0.25 mM dNTPs (MD Bio.), 20 U of RNasin I Plus RNase Inhibitor (Promega, San Luis Obispo, CA, USA) and 20 U of M-MLV Reverse Transcriptase (Promega) and incubated at 42 °C for 90 min to allow for cDNA synthesis. This mixture was then used for specific cDNA amplification in a GeneAmp PCR system 2400 (Perkin Elmer, Waltham, MA, USA).