Figure 8. Autophagy restimulation requires mRNA preaccumulation and is amplified by NEDD4L down-regulation. (A) HeLa cells were treated with EBSS for the indicated time periods. After 6 h, in some samples, full medium was added for 15 min, and then cells were starved again for 1 and 2 h, respectively. Protein levels of ULK1 and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (below graph). Data are expressed as the mean ± SEM (n = 3). (B) HeLa cells were treated with EBSS as in A. In some samples, Act D was added for the first 4 h of starvation. Then, cells were analyzed both by WB and by immunofluorescence. Protein levels of ULK1 and ACTIN were detected by WB. ATG16L puncta occurrence (red puncta) was visualized by confocal microscopy. Analysis of the number of ATG16L puncta per cell is shown in the graph. Data are expressed as the mean ± SEM (n = 3); representative images are shown. Bar, 20 µm (>50 cells analyzed per sample). (C) NEDD4L expression was down-regulated in HeLa cells by using specific RNAi oligos (siRNA NEDD4L#1) or unrelated oligos as a negative control (siRNA CTRL). Cells were nutrient-starved as in A. Protein extracts were next analyzed by WB for the expression of ULK1, NEDD4L, and ACTIN. ATG16L puncta occurrence (red puncta) was visualized by confocal microscopy. Analysis of the number of ATG16L puncta per cell is shown in the graph. Data are expressed as the mean ± SEM (n = 3); representative images are shown. Bar, 20 µm (>50 cells analyzed per sample). Data represent mean ± SEM (n = 3). In A, data were analyzed by one-way ANOVA followed by Tukey post hoc test. **, P < 0.01. In B and C, data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Figure 8.

Autophagy restimulation requires mRNA preaccumulation and is amplified by NEDD4L down-regulation. (A) HeLa cells were treated with EBSS for the indicated time periods. After 6 h, in some samples, full medium was added for 15 min, and then cells were starved again for 1 and 2 h, respectively. Protein levels of ULK1 and ACTIN were detected by WB. Densitometric analysis of ULK1 over ACTIN is also shown (below graph). Data are expressed as the mean ± SEM (n = 3). (B) HeLa cells were treated with EBSS as in A. In some samples, Act D was added for the first 4 h of starvation. Then, cells were analyzed both by WB and by immunofluorescence. Protein levels of ULK1 and ACTIN were detected by WB. ATG16L puncta occurrence (red puncta) was visualized by confocal microscopy. Analysis of the number of ATG16L puncta per cell is shown in the graph. Data are expressed as the mean ± SEM (n = 3); representative images are shown. Bar, 20 µm (>50 cells analyzed per sample). (C) NEDD4L expression was down-regulated in HeLa cells by using specific RNAi oligos (siRNA NEDD4L#1) or unrelated oligos as a negative control (siRNA CTRL). Cells were nutrient-starved as in A. Protein extracts were next analyzed by WB for the expression of ULK1, NEDD4L, and ACTIN. ATG16L puncta occurrence (red puncta) was visualized by confocal microscopy. Analysis of the number of ATG16L puncta per cell is shown in the graph. Data are expressed as the mean ± SEM (n = 3); representative images are shown. Bar, 20 µm (>50 cells analyzed per sample). Data represent mean ± SEM (n = 3). In A, data were analyzed by one-way ANOVA followed by Tukey post hoc test. **, P < 0.01. In B and C, data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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