Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet: The observed ATG5-c-Myc regulatory axis is involved in the regulation of mouse embryonic stem cell (ESC) differentiation (A) An Oct4-EGFP mouse ES (Oct4-EGFP-mES) cell line was used to establish the ESC differentiation system. Briefly, ESCs were cultured for the indicated durations in differentiation medium from which LIF (leukemia inhibitory factor) was removed and to which RA (retinoic acid, 1 μM) was added. The differentiation efficiency was determined by analyzing the morphologies of the colonies (upper left), the expression of Oct4-regulated EGFP (upper right), and the staining of AP (alkaline phosphatase) (lower). (B) Oct4-EGFP-mESCs were differentiated for 4 days as described in (A). IF analyses (left) were then performed to determine the levels of LC3 (red) and GFP-Oct4 (green). DAPI staining indicates the nucleolus (blue). Western blot analyses were also performed with antibodies as indicated (right). (C) Oct4-EGFP-mESCs were differentiated as described in (A). Western blot and RT-PCR analyses were performed with antibodies as indicated. (D) Oct4-EGFP-mESCs were differentiated for 4 days as described in (A). Cells were then treated with 25 μM MG132 for 6 h. Western blot analyses were performed with antibodies as indicated. (E) Oct4-EGFP-mESCs were stably transfected with an ATG5-specific shRNA or a control shRNA. Cells were then differentiated as described in A by removing LIF and adding RA. Western blot analyses were performed with antibodies as indicated. (F) Oct4-EGFP-mESCs were differentiated as described in (A). Cells were then treated with 25 μM MG132 for 6 h. Co-IP assays with antibodies against ATG5 were performed. IgG antibodies were used as a negative control. (G) Oct4-EGFP-mESCs were stably transfected with an ATG5-specific shRNA, an ATG5-specific shRNA combined with a c-Myc-specific shRNA, or a control shRNA as indicated. Cells were then differentiated for 4 days as described in A by removing LIF and adding RA. AP staining assays were performed to determine the differentiation efficiency.

Article Snippet: OG2 mouse embryonic stem cells (ESCs) , The Jackson Laboratory , 004654.

Techniques: Cell Culture, Expressing, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, shRNA, Control, Co-Immunoprecipitation Assay, Negative Control

Journal: iScience

Article Title: A nonautophagic role of ATG5 in regulating cell growth by targeting c-Myc for proteasome-mediated degradation

doi: 10.1016/j.isci.2021.103296

Figure Lengend Snippet:

Article Snippet: OG2 mouse embryonic stem cells (ESCs) , The Jackson Laboratory , 004654.

Techniques: Virus, shRNA, Recombinant, Plasmid Preparation, Software, Luciferase, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription