*2.4. Gelatin Zymography Protease Assay*

The activities of MMP-2 and MMP-9 in the serum-free conditioned medium were evaluated by gelatin zymography according to a previously described method by Huang et al. [22]. In short, samples were prepared with standard sodium dodecyl sulfate (SDS)-gel loading buffer containing 0.01% SDS (Sigma-Aldrich, St Louis, MO, USA). The prepared samples (25 μg total protein) were not boiled before loading, but subjected to electrophoresis on 8% SDS polyacrylamide gels (1.0-mm-thick, acrylamide/bis-acrylamide = 30/1.2) containing 0.1% gelatin (Sigma-Aldrich, St Louis, MO, USA). After electrophoresis, the gel was washed twice with 100 mL distilled water containing 2% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) on a shaker for 30 min at room temperature to remove SDS, and incubated in 100 mL reaction buffer (0.02% NaN3, 10 mM CaCl2 and 40 mM Tris-HCl (pH 8.0)) at 37 ◦C for 12 h. The gel was further stained with Coomassie brilliant blue R-250 dye (Abcam plc, Cambridge, UK) followed by destaining with methanol/acetic acid/water (50:75:875, *v*/*v*/*v*).

#### *2.5. Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)*

Total RNAs were extracted using a TRIzol reagen<sup>t</sup> (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, as described by Chiu et al. [18]. In general, the mRNA levels were analyzed by quantitative real-time RT-PCR using a Bio-Rad iCycler system (Bio-Rad, Hercules, CA, USA), and normalized to the housekeeping gene, β-actin. The sequences of primers (MDBio Inc., Taipei, Taiwan) used in the experiments are listed in Table S3.
