Fig. 3

Halofuginone induces autophagosome formation and eventually interferes with its fusion with lysosomes. a WRO cells were transiently transfected with ATG7 siRNA or control duplex (Ct. Du.) siRNA. After 36 h, the cells were exposed to 100 nM halofuginone (HF) for 8 h. The expression of ATG7, LC3B and β-Tubulin was analyzed by immunoblotting of cell homogenates. Representative immunoblots are shown along with LC3B-II/I band intensity ratios as index of autophagosome formation. b WRO cells plated on coverslips were treated with 100 nM halofuginone (HF) in the presence or absence of 10 mM ammonium chloride (NH₄⁺). After 8 h the cells were fixed, processed for LC3 (green) and LAMP-1 (red) immunostaining and imaged by fluorescence microscopy. Nuclei were stained with DAPI. Scale bars: 10 μm. c Bars indicate the average yellow fluorescence intensity density of immunofluorescences shown in b. Data are from 5 different images for each condition. Error bars: standard deviation. Statistically significant differences between fluorescence intensity densities before and after NH₄⁺ are shown (*, p ≤ 0.05). The images shown are representative of four separate experiments. d WRO cells were plated on coverslips and transiently transfected with vectors expressing GFP-FYVE or GFP-LC3. After 36 h, the cells were exposed to 100 nM halofuginone (HF) for 8 h. Following HF, cells were imaged by fluorescence microscopy. Scale bars: 10 μm