Figure 4.

ATG5 interacts with c-Myc under normal culture conditions

(A) 293T cells were transfected with a Flag-tagged ATG5 and an HA-tagged c-Myc constructs for 48 h. Cells were lysed and subjected to coimmunoprecipitation (co-IP) assays with antibodies against Flag (left) or HA (right) as indicated.

(B) Co-IP assays were performed in 293T cells with antibodies against ATG5 (left) or c-Myc (right) to determine the interaction between endogenous ATG5 and c-Myc proteins.

(C) 293T cells were transfected with a GFP-tagged ATG5 and an HA-tagged c-Myc construct for 48 h under normal or serum starvation conditions. Immunofluorescence (IF) analysis was then performed to evaluate the localization of ATG5 (green) and c-Myc (red). The blue signal indicates the nucleolus (stained with DAPI).

(D) GST-tagged ATG5 and His-tagged c-Myc were expressed in E coli and purified (left two panels). The two proteins were co-incubated, and IP assays with antibodies against ATG5 or c-Myc (right two panels) were performed as indicated to evaluate the direct binding between ATG5 and c-Myc in vitro.

(E) 293T cells co-transfected with a GFP-tagged ATG5 and an HA-tagged c-Myc were further cultured under normal conditions or serum starvation conditions as indicated. Cell extracts were prepared, and co-IP assays with antibodies against GFP were performed. Co-IP with IgG antibodies was performed as a negative control.

(F) 293T cells transfected with a flag-tagged ATG5 and a GFP-tagged LC3 were cultured under normal conditions or serum starvation conditions as indicated. IF analysis was performed to evaluate the localization of ATG5 (red) and LC3 (green). DAPI staining was used to indicate the nucleolus (blue).

(G) Molecular model. ATG5 interacts with c-Myc and promotes c-Myc protein degradation under normal culture conditions. However, when cells are subjected to serum starvation to activate autophagy, ATG5 may be released from c-Myc and mainly contribute to autophagy.