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. 2021 Oct 16;24(11):103296. doi: 10.1016/j.isci.2021.103296

Figure 2.

Figure 2

ATG5 negatively regulates the protein level of c-Myc, especially under normal culture conditions

(A) HeLa cells were transfected with an ATG5-specific siRNA or a Flag-tagged ATG5 construct and were further cultured under normal conditions or serum starvation conditions for 48 h. Western blot analyses or RT-PCR analyses were then performed.

(B) 293T cells were transfected with an ATG5-specific siRNA or a Flag-tagged ATG5 construct and were further cultured under normal conditions or serum starvation conditions for 48 h. Western blot analyses were then performed.

(C) Flag-tagged ATG5 or ATG5-specific siRNA-transfected 293T cells were further transfected with a control luciferase reporter construct or a c-Myc-targeted luciferase reporter construct as indicated. Cells were then cultured under normal conditions or serum starvation conditions for 48 h. Luciferase activity was evaluated.

(D) 293T cells were transfected with an ATG5-specific siRNA or a Flag-tagged ATG5 construct and were further cultured under normal conditions or serum starvation conditions for 48 h as indicated. Cells were then lysed and subjected to quantitative RT-PCR analyses.

(E) HeLa cells transfected with a Flag control construct, a Flag-tagged ATG5 construct, or a Flag-tagged ATG5 construct together with an HA-tagged c-Myc construct were cultured under normal conditions for the indicated durations. The relative cell growth ability was examined by CCK-8 assays.

(F) HeLa cells were transfected with 3 individual c-Myc-specific siRNAs for 48 h. Western blot analyses were performed to determine the knockdown efficiency of the c-Myc siRNAs (left). HeLa cells transfected with a control siRNA, an ATG5-specific siRNA, or an ATG5-specific siRNA together with a c-Myc-specific siRNA as indicated were cultured under normal conditions for the indicated durations. The relative cell growth ability was examined by CCK-8 assays (right).

(G) Predicted model. Under normal culture conditions, ATG5 negatively regulates c-Myc and further affects the expression of c-Myc downstream target genes and cell growth. Statistics: all experiments were repeated more than 3 times. The bars and error bars indicate the mean ± s.d. values; n = 3 independent replicates. Two-tailed unpaired Student's t test was performed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.