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Figure 5 | Molecular Cancer

Figure 5

From: ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy

Figure 5

Genotoxic stress-induced autophagy and autophagy flux is modulated by ISG20L1. (a) U2OS cells were reverse transfected with three unique siRNAs targeting ISG20L1 and treated for 24 h (0.13 mM 5-FU) to induce autophagy as seen in the siControl lane by the modification of LC3-I to LC3-II. Western blot analysis is representative of four independent experiments. (b) U2OS cells were reverse transfected with control or siRNA targeting ISG20L1 and 3 days later treated for 24 h with 5-FU. Electron microscopy was performed and representative images can be seen (left panel) Arrowheads indicate autophagosomes or autophagolysosomes. Morphometric analyses was performed on electron micrographs and the percentage of autophagic vacuoles per cytoplasmic volume is shown. Results represent the mean and standard error, p < .0001, n = 25 (right panel). This experiment was performed in duplicate. (c) Vector control and ISG20L1 were transfected in U2OS cells stably expressing the tandemly tagged mRFP-GFP-LC3 (tLC3) and then treated for 24 h with 5-FU. Representative panels show GFP alone and RFP alone for control and ISG20L1 as well as magnification of the area outlined by the white box of ISG20L1. White arrows highlight RFP+GFP- only foci representative of autolysosomes (late stage autophagy, RFP+GFP-); and yellow arrows indicate early autophagosomes where both GFP and RFP are fluorescing (RFP+GFP+). Quantitation was performed and results are the mean and standard error of RFP+GFP- foci expressed as a percentage of total foci (p < .001, n = 50).

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