Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious brokers. CK cells isolated from SPF Rhode Island Red chicks and recombinant adenoviruses expressing the three EGFP-Av-LC3 and FLAG-mCherry-Av-LC3 paralogs, as well as EGFP-Av-LC3B made up of a G120A mutation, were generated, as explained in FRFRFRRRRG120A FG120A RGGAAGATTGCACnucleotide sequence accession numbers used were “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_417327.2″,”term_id”:”118100496″,”term_text”:”XM_417327.2″XM_417327.2, “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001031461″,”term_id”:”71895448″,”term_text”:”NM_001031461″NM_001031461 and “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_419549″,”term_id”:”1390103007″,”term_text”:”XM_419549″XM_419549. Avian EGFP-LC3 paralogs respond to treatments to induce or inhibit autophagy The power of the three avian EGFP-LC3 paralogs expressed from your recombinant adenovirus vectors in studying autophagic signaling was investigated initially in main CK cells. Cells were transduced with either a control adenovirus expressing GFP, rAd5-GFP (kindly provided by Sharon Tooze10) or rAd5-EGFP-Av-LC3A, -LC3B or Rabbit Polyclonal to SREBP-1 (phospho-Ser439) -LC3C. Twenty-four hours post-transduction, RAD001 inhibitor cells were incubated for 2 h under conditions known to induce or inhibit autophagy. Transduced control cells incubated in full-nutrient medium (FM) were compared RAD001 inhibitor with transduced cells incubated in either HBSS, to induce autophagy, or in HBSS with 10 nM wortmannin, a PI3 kinase/PtdIns 3-kinase inhibitor known to inhibit autophagosome formation. Confocal microscopy analysis revealed that GFP, expressed from rAd5-GFP, experienced a diffuse distribution throughout the cytoplasm and the nucleus of the CK cells and that distribution was unaffected by any of the treatments (Fig.?2A). Similarly, when CK cells were transduced with rAd5-EGFP-Av-LC3A or -LC3B and incubated in full-nutrient media the EGFP-Av-LC3 transmission was observed to be distributed diffusely throughout the cytoplasm and the nucleus (Fig.?2A). When CK cells were transduced with rAd-EGFP-Av-LC3C and incubated in full-nutrient media, the EGFP-Av-LC3C distribution was observed to be diffuse in the cytoplasm and the nucleus, but there was also some reticular localization (Fig.?2A). However, when the CK cells were starved in HBSS, EGFP-Av-LC3A, -LC3B and -LC3C proteins were relocated to unique cytoplasmic puncta, suggestive of autophagosome formation (Fig.?2A). As can be observed from Physique?2A, the puncta associated with autophagosome formation did not form in starved cells in the presence of wortmannin, showing that, as seen for autophagy induction in other animal systems, formation of EGFP-LC3 puncta was dependent on class III PtdIns3K activity. Finally, as shown in Physique?2B, for EGFP-Av-LC3A, EGFP-Av-LC3B and EGFP-Av-LC3C there was a significant increase in the number of puncta per cell in CK cells starved in HBSS when compared with those incubated in FM and that this increase in puncta number was inhibited in the presence of wortmannin. There was no switch in the number of puncta per cell in CK cells expressing GFP from rAd5-GFP. Open in a separate window Physique?2. EGFP-Av-LC3 paralogs are markers for autophagy in main chick kidney (CK) cells, chick embryo fibroblasts (CEFs) and DF1 cells. (A) CK cells were transduced with rAd-EGFP-Av-LC3A, rAd-EGFP-Av-LC3B, rAd-EGFP-Av-LC3C RAD001 inhibitor or rAd-GFP. After 24 h cells were washed and incubated in full media (FM; control), Hanks balanced salt answer (HBSS; to induce autophagy) or HBSS made up of 10 nM wortmannin (HBSS+WM; to inhibit HBSS-induced autophagy). Nuclei (blue) were stained with DAPI. Level bars: 10 m. (B) Quantity of puncta per cell was decided from (A) using Imaris spots software. (C) CEF cells were treated as in (A) and puncta per cell decided as in (B). (D) DF1 cells were treated as in (A) and puncta per cell decided as in (B). Open bars, FM treated cells; closed bars, HBSS treated cells; hashed bars, HBSS+WM treated cells. (E) CK cells were transduced with rAd-EGFP-Av-LC3B or rAd-EGFP-Av-LC3B G120A. After 24 h cells were washed and incubated in HBSS. Nuclei were stained with DAPI. Level bars: 10 m. (F) Quantity of puncta per cell was decided from (E) using Imaris spots software. Mean RAD001 inhibitor plus standard deviation is shown for at least 20 cells counted from three impartial experiments. **p.