Autophagy Suppresses Toll-Like Receptor 3-Mediated Inflammatory Reaction in Human Epidermal Keratinocytes

Figure 4

(a) Keratinocytes were transduced with recombinant adenovirus expressing green fluorescent protein-light chain 3 (GFP-LC3) overnight. After changing the medium, the cells were further incubated with fresh growth medium for 1 day. The cells were then treated with 1 μg/mL of poly (I:C) and 3-methyladenine (3-MA) (5 μM). Autophagic LC3 puncta were observed under fluorescent microscopy. Cotreatment with 3-MA inhibited poly (I:C)-induced autophagy. (b) The cells were cotreated with poly (I:C) and 3-MA for 2 h. The messenger RNA (mRNA) level for inflammatory cytokines was determined by real-time polymerase chain reaction. The 3-MA cotreatment enhanced poly (I:C)-induced gene expression of inflammatory cytokines. (c) The cells were cotreated with poly (I:C) and 3-MA for 24 h. The conditioned media were collected, and secreted inflammatory cytokines were determined by enzyme-linked immunosorbent assay. The 3-MA cotreatment enhanced poly (I:C)-induced secretion of inflammatory cytokines. (d) Effect of 3-MA on inflammasome-related molecules.

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