Figure 7

FK866-induced autophagy is associated with JNK signaling in primary hepatocytes. Hepatocytes were treated with SP600125 (SP, 20 µmol/L, 30 min) or transfected with Jnk siRNA (100 nmol/L, 48 h) prior to GaIN/LPS (G/L, 1 mg/mL /30 ng/mL, 24 h) challenge. (a) JNK activity was examined by western blot. The data are shown as the means ± SEM of three independent experiments performed in duplicate. *P < 0.05 indicates significant differences. (b) The protein expression levels of p-JNK, JNK, and autophagy indicators are shown as densitometric graphs of the optical density-based data of immunoblot. The data are shown as the means ± SEM of three independent experiments. *P < 0.05. (c) Representative fluorescence micrographs showed autophagy vacuoles in hepatocytes with G/L in the presence of SP or vehicle (Veh) from a pool of at least 10 images. Original magnification × 200, scale bar 50 µm. (d) Quantification of autophagosomes and autolysosomes. *P < 0.05. (e) Measurement of LDH release of primary hepatocytes. The data are shown as the means ± SEM of three independent experiments. *P < 0.05 vs. the corresponding Veh group. ^# P < 0.05 vs. the corresponding control group. (f) JNK protein expression was effectively suppressed using Jnk-specific siRNA in hepatocytes. (g) Western blot analysis of JNK, ATG7, p62, and LC3B expression in the presence or absence of Jnk siRNA. The data are shown as the means ± SEM of three independent experiments. *P < 0.05. (h) Determination of LDH levels in the cultured medium. The data are shown as the means ± SEM of three independent experiments. *P < 0.05 vs. the corresponding Control siRNA group. ^# P < 0.05 vs. the corresponding Veh group.