Vandetanib

A GFP-tagged LC3 expression vector has recently been utilized to detect autophagy. Calu-6 cells were seeded (2 × 10⁵ cells/well) into 12-well plates overnight. GFP-LC3-expressing plasmids were then transiently transfected into the cells with Lipofectamine™ 2000 (Invitrogen, Life Technologies, Cat.11668-019) according to the manufacturer's instructions. At 24 h after transfection, the cells were treated with vandetanib and NAC (Beyotime, Cat.S0077). At the end of the treatment period, autophagy was examined by counting the percentage of cells with GFP-LC3 puncta using a confocal microscopy. We counted a minimum of 200 cells per sample, and each experiment was performed in triplicate.

At E13, the eggs in the incubator were candled to remove unfertilized eggs and dead embryos. Embryonic eggs from the layer lineage were randomly selected for intra-ovo injection. Briefly, a small hole was drilled at the wide end of the egg after cleaning the eggshell with 75% alcohol. Vandetanib (Beyotime Biotechnology, Shanghai, China) diluted in PBS (75 μg/mL) was injected into the air chamber through the hole without passing through the chamber, with a dose equivalent to 15 μg per egg. PBS-injected eggs were used as control. After the inoculations were completed, the hole was sealed with paraffin solution and the eggs were placed back into the same incubator to allow them to continue developing.