Figure 4.

Expression of the autophagy-related protein LC3B in thyroid tissues of Hashimoto’s thyroiditis (HT) patients and the effect of interleukin-23 (IL-23) on autophagy activity in Nthy-ori 3-1 cells. (A) The levels of the autophagy-related protein LC3B in thyroid tissues from HT patients (n = 10) and healthy controls (n = 5) were examined with immunohistochemistry (IHC) analysis. The brown regions represent positive expression. The slides were analyzed under a 400× microscope equipped with a camera (scale bars, 50 µm). (B) Cumulative IHC quantification data from all samples are shown. Significant differences and P values were calculated with the Mann–Whitney U test. ***P < 0.001 vs. controls. (C) Nthy-ori 3-1 cells were treated with IL-23 (50 ng/mL) at different time points, the LC3B-I/II expression levels were measured by western blot. (D) The change of LC3B-II expression in Nthy-ori 3-1 cells was analyzed at 3 h by western blot in the presence of IL-23 (50 ng/mL) and/or anti-IL-23 pretreatment (5 µg/mL). (E) Nthy-ori 3-1 cells were transfected with mRFP-GFP-LC3, and then were treated with IL-23 (50 ng/mL) and/or anti-IL-23 pre-treatment (5 µg/mL). Representative images of the fluorescent LC3 puncta are shown (1,000×; scale bars, 5 µm). (F) Mean number of autophagosomes (puncta with yellow color in the merged images) and autolysosomes (puncta with only red color in the merged images) per cell were analyzed. The results shown are representative of three replicates. Significant differences and P values are calculated with the Kruskal–Wallis H test. **P < 0.01, ***P < 0.001.