Figure 4.

Serum amyloid A (SAA) induction of interleukin (IL)-1β is via NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in keratinocytes at post-transcriptional level. (a) The neonatal foreskin-isolated normal primary kerationocytes were stimulated with SAA (10 μg/ml) for 24 h, and the mRNA expression of caspase-1 and NLRP3 was determined using real-time transcription–polymerase chain reaction (RT–PCR) and normalized against the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Gene expression is graphed as mean fold induction over medium control. The data represent the mean ± standard error of the mean (s.e.m.) of three independent experiments (**P < 0·01). (b) Normal primary keratinocytes were treated with indicated concentrations of SAA (1 and 10 μg/ml) for 24 h. The cells were lysed and the whole-cell extracts were assessed by Western blot using antibodies against NLRP3 (118 kDa) and the mature form of caspase-1 (20 kDa). Representative blots of three independent experiments are shown. (c) Normal primary keratinocytes were treated with caspase-1 inhibitor Z-YVAD-fmk at the indicated concentrations (1 and 10 μg/ml) for 30 min and then stimulated with SAA (10 μg/ml). Culture supernatants were collected after 24 h and assayed with IL-1β enzyme-linked immunosorbent assay (ELISA). The data represent the mean ± s.e.m. of three experiments with similar results (*P < 0·05; **P < 0·01). Keratinocytes were transfected with siRNA oligonucleotides specific for NLRP3 (siNLRP3) or a non-specific siRNA oligonucleotide (siControl), and subsequently stimulated with SAA (10 μg/ml) for 24 h, and the levels of NLRP3 mRNA (d) and protein (e) were analysed using RT–PCR and Western blot. One experiment representative of three experiments. (f) siRNA-transfected keratinocytes were then stimulated with SAA (10 μg/ml), and culture supernatants were collected after 24 h and assayed with IL-1β ELISA. The data represent the mean ± s.e.m. of three experiments with similar results (**P < 0·01).