Figure 4

ISG20L1 expression modulates autophagy. (a) RKO cells were transfected with control, pCEP4, or ISG20L1 for 24 h before harvesting for Western analysis of ISG20L1, LC3, and actin. The Western blot is representative of four independent experiments. (b) RKO cells were reverse transfected with control or siISG20L1 and then treated with etoposide (20 μM) for 8 h before harvesting for Western analysis of ISG20L1, LC3, and actin. Western blot is representative of five independent experiments. (c) As performed in (b) knockdown of ISG20L1 suppresses autophagy as measured by IHC using an antibody specific to the cleaved form of LC3. IHC analysis of LC3 was performed in RKO cells induced to undergo autophagy after treatment with etoposide (20 μM). Controls include no siRNA and transfection of siRNA alone. (d) Results from (c) were quantified by counting number of cells staining positive for LC3 (dark brown) and dividing by the total number of cells (purple nuclear stain) to attain % LC3. Results are representative of 3 independent experiments and error bars represent standard deviation. (e) To measure autophagy flux, RKO cells were reverse transfected with control or siISG20L1 and treated with etoposide (20 μM) and lysosomal inhibitors (10 μg/mL E64d and pepstatin A) for 8 h before harvesting for Western analysis of ISG20L1, LC3, and actin. Results are representative of four independent experiments.