Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

Figure 7

Downregulation of cytokine and chemokine release by ADAM8 knockdown. (a–f) The indicated types of human liver cells were left untransduced or transduced with lentiviruses coding for two different sequences of shRNA against ADAM8 expression (A8KD1 and A8KD2) or with control shRNA (Ctrl) to knockdown the ADAM8 expression. After 72 h, knockdown of ADAM8 was controlled on mRNA (a–c) and protein level (d–f). ADAM8 mRNA expression was quantified by qPCR and normalised to the expression of GAPDH for human cells. ADAM8 protein was detected by Western blot analysis and shown as representative blot for each cell line (d–f). The band intensities were quantified by densitometry and normalised to that of GAPDH. (g–l) The indicated types of cells with or without ADAM8 knockdown were left unstimulated (US) or stimulated with the indicated mediators. After 24 h, concentrations of released cytokines (TNF-α and IL-6) and chemokines (CX3CL1) in the supernatants were determined by ELISA. Quantitative data are shown as the of 3-4 independent experiments, and representative Western blots are shown. Significant differences caused by cell stimulation are indicated by asterisks (, , and ), and differences caused by knockdown of ADAM8 compared to control are indicated by hashtags (^#, ^##, and ^###).