Image quant software program

PMA-primed THP-1 cells (1×10⁷) were treated with 100 µg/mL of silver materials for 24 h and then, all RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen, Germany) in accordance with the manufacturer's protocol. Extracted RNA was reverse transcribed using the Reverse Transcription System (Promega, USA). Synthesized cDNA was amplified by PCR using Taq polymerase (GeneAll, Korea). The sequence of specific primers for NALP3 (cytoplasmic receptor) and GAPDH was as follows: NALP3 forward: 5′-TGCCTTTGACGAGCACATAG-3′; NALP3 reverse: 5′-GCAGCAAACTGG AAGGAAG-3′; caspase-1 forward: 5′-GAAGGCATTTGTGGGAAGAA-3′; caspase-1 reverse: 5′-CATCTGGCTGCTCAAATGAA-3′; and GAPDH forward: 5′-GAGTCAACGGATTTGGTCGT-3′; GAPDH reverse: 5′-TTGATTTTGGAGGGATCTCG-3′. After 5 min at 95°C, 28 cycles at 95°C for 1 min, 53°C for 1 min (caspase-1; 51°C), and 72°C for 2 min were performed, ending with a final extension step at 72°C for 5 min. Quantification of PCR products was performed by electrophoresis. Data was analyzed using the Image Quant software program (GE Healthcare, USA).

Protein extracts were separated by one dimension polyacrylamide gel electrophoresis 12% (SDS-PAGE) with molecular weight markers (S1 Fig) in a side lane, transferred to nitrocellulose membranes for 1 hour and 30 minutes and blocked with skim milk in TBS-Tween for one hour. For collagen-VI detection, the proteins were transferred in a wet-system overnight at 4°C. The proteins were probed against specific antibodies to vimentin (ab45939, 1:2000, Abcam, San Francisco, CA), lumican(sc-166871, 1:2000, Santa Cruz, Heidelberg, Germany), collagen-VI alpha chain (sc-20649, 1:2000, Santa Cruz, Heidelberg, Germany), and vitronectin (sc-7776, 1:1000, Santa Cruz, Heidelberg, Germany).The expression level of beta-actin (ab8226, 1:5000, Abcam, San Francisco, CA, USA) was used to control for equal loading.
Densitometry analysis of immunoblots of all proteins mentioned above was performed using the Image Quant software program purchased from GE Healthcare, Upsalla, Sweden. Relative protein expression was calculated as pixel volume in specific band/pixel volume in beta-actin band for each lane.