Figure 7. ULK1 restoration requires de novo mTOR-dependent protein synthesis. (A) HeLa cells were treated with EBSS for the indicated time periods. The levels of p-mTOR, mTOR, ULK1, p-ULK1 S757, ULK1, and ACTIN were detected by WB. Densitometric analysis of p-mTOR over ACTIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 4). (B) HeLa cells were treated with EBSS for the indicated time periods; at 4 h of starvation, rapamycin (100 nM) was added. Levels of p-mTOR, mTOR, ULK1, p-ULK1 S757, and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (right graph). Data are expressed as the mean ± SD (n = 3), and statistical analysis was performed using unpaired Student's t test. *, P < 0.05. (C) HeLa cells were treated with EBSS for the indicated time periods and at 4 h of starvation, 250 nM Torin1 or 50 µM CHX was added. Levels of p-mTOR, mTOR, p-p70S6K, ULK1, and ACTIN were detected by WB. (D) HeLa cells were treated with EBSS for 6 h in the presence or not of Act D. Levels of ULK1 and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (right graph). Data are expressed as the mean ± SD (n = 3) and statistical analysis was performed using unpaired Student's t test. *, P < 0.05. (E) qPCR analysis of polysomal recruitment of ULK1 and ACTIN mRNAs in control (CTRL) and EBSS-starved (2 h EBSS and 6 h EBSS) HeLa cells. Densitometric analysis of the signal in each fraction was performed, and the results were represented as the percentage of total signal in all fractions. Data represent mean ± SEM (n = 3). (F) NEDD4L expression was down-regulated by RNAi. Cells were nutrient-starved for 12 h in the presence or not of Baf A1 and analyzed by MTS assay. NEDD4L down-regulation was detected by WB. In A and C, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. In E and F, data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison post test. ***, P < 0.001; ****, P < 0.0001.
Figure 7.

ULK1 restoration requires de novo mTOR-dependent protein synthesis. (A) HeLa cells were treated with EBSS for the indicated time periods. The levels of p-mTOR, mTOR, ULK1, p-ULK1 S757, ULK1, and ACTIN were detected by WB. Densitometric analysis of p-mTOR over ACTIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 4). (B) HeLa cells were treated with EBSS for the indicated time periods; at 4 h of starvation, rapamycin (100 nM) was added. Levels of p-mTOR, mTOR, ULK1, p-ULK1 S757, and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (right graph). Data are expressed as the mean ± SD (n = 3), and statistical analysis was performed using unpaired Student's t test. *, P < 0.05. (C) HeLa cells were treated with EBSS for the indicated time periods and at 4 h of starvation, 250 nM Torin1 or 50 µM CHX was added. Levels of p-mTOR, mTOR, p-p70S6K, ULK1, and ACTIN were detected by WB. (D) HeLa cells were treated with EBSS for 6 h in the presence or not of Act D. Levels of ULK1 and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (right graph). Data are expressed as the mean ± SD (n = 3) and statistical analysis was performed using unpaired Student's t test. *, P < 0.05. (E) qPCR analysis of polysomal recruitment of ULK1 and ACTIN mRNAs in control (CTRL) and EBSS-starved (2 h EBSS and 6 h EBSS) HeLa cells. Densitometric analysis of the signal in each fraction was performed, and the results were represented as the percentage of total signal in all fractions. Data represent mean ± SEM (n = 3). (F) NEDD4L expression was down-regulated by RNAi. Cells were nutrient-starved for 12 h in the presence or not of Baf A1 and analyzed by MTS assay. NEDD4L down-regulation was detected by WB. In A and C, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. In E and F, data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison post test. ***, P < 0.001; ****, P < 0.0001.

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