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. 2019 Nov 7;20(22):5543. doi: 10.3390/ijms20225543

Figure 5.

Figure 5

Silencing of ULK1 arrests autophagy induction upon rapamycin treatment. ULK1 was silenced by siRNA in HEK293T cells, followed by 100 nM rapamycin treatment in time. (A) Test of the ULK1 silencing by immunoblotting, total level of ULK1 and ULK1-Ser555-P were followed. GAPDH was used as the loading control. Densitometry data represent the intensity of ULK1 and ULK1-Ser555-P normalized for GAPDH and ULK1-Ser555-P normalized for total level of ULK1. (B) The markers of autophagy (LC3, p62), AMPK, and mTOR (p70S6K-P, 4E-BP1-P) were followed by immunoblotting. GAPDH was used as the loading control. (C) Densitometry data represent the intensity of p62 and LC3 II normalized for GAPDH, p70S6K-P normalized for the total level of p70S6K, 4E-BP1-P normalized for the total level of 4E-BP1, and AMPK-Thr172-P normalized for the total level of AMPK. For each of the experiments, three independent measurements were carried out. Error bars represent standard deviation, asterisks indicate statistically significant difference from the control: ns—nonsignificant; * p < 0.05; ** p < 0.01. (D) Computer simulation of rapamycin treatment. The relative activity of AMPK-Thr172-P, mTOR, ULK1-Ser555-P and autophagy (ATG) are plotted in time.