Fig. 3.

Effects of ApN, miR-711 (a) and FADD (b) on NLRP3 expression in vitro. a C2C12 myotubes were either treated or not with ApN or transfected with miR-711 mimic or its control (Ctrl+) for 24 h. Cells were also transfected with anti-miR-711 or its relative control (Ctrl–) for 28 h, while ApN was added during the last 24 h. All conditions presented herein were obtained in C2C12 challenged by LPS except for the basal condition (no LPS, no transfection or any other treatments) represented by the dotted line. mRNA levels were normalized to cyclophilin, and the subsequent ratios were presented as relative expression compared to basal condition. Values are means ± SEM for seven repeated experiments. A Western blot with five experimental conditions (basal, LPS alone, ApN + LPS, control miR + LPS and miR mimic + LPS) is also shown above the histograms. NLRP3 protein expression was normalized to actin levels and the subsequent ratios presented as relative expression compared to basal condition. This blot is representative of two different experiments; the mean NLRP3 value is indicated at the top of each condition. b Effects of FADD silencing on NLRP3 expression in C2C12 cells challenged by LPS. Myotubes were transfected for 24 h with small interfering RNAs (siRNAs) against FADD or a negative (non-targeting, NT) control. Next, cells were challenged by LPS during the last 20 h. mRNA levels were normalized and presented as relative expression compared to the basal condition (no LPS, no transfection) represented by the dotted line. NLRP3 protein expression was measured by Western blot, normalized to actin levels, and also presented as percentages of the basal condition. Values are means ± SEM for five (mRNA) and three (protein) repeated experiments. **P < 0.01, ***P < 0.001 for indicated conditions (a) or vs NT siRNA (b)