Figure 1.

OTULIN is essential for autophagosome formation. (A) The number of LC3B-positive puncta is less in OTULIN-overexpressing cells under starvation conditions. HeLa cells stably expressing mRFP-EGFP-LC3B were transfected with either the vector control or Flag-OTULIN or Flag-OTULIN^C129S and treated with EBSS for 4 h. Cells were fixed and stained with DAPI (blue) and representative fluorescence images of LC3B puncta are shown. Scale bar: 5 μm. (B) Quantification of yellow (RFP⁺GFP⁺) and red (RFP⁺GFP⁻) puncta per cell as represented in Figure 1A and S1A. Data are mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA). (C) Depletion of OTULIN induces the aggregation of the LC3B-positive puncta in cells under starvation conditions. HeLa cells stably expressing mRFP-EGFP-LC3B were transfected with either the control shRNA or OTULIN shRNA-1 or shRNA-2, and treated with EBSS for 4 h. Cells were fixed and stained with DAPI (blue) and representative fluorescence images of OTULIN shRNA-1 are shown. The enlarged images of the white square part under each condition are shown in the following pictures. Scale bar: 5 μm. (D) Quantification of yellow (RFP⁺GFP⁺) and red (RFP⁺GFP⁻) puncta per cell as represented in Figure 1C and S1B. Data are mean ± SD from three independent experiments. ***p < 0.001 (one-way ANOVA). (E) Western blot analysis of the knockdown efficiency of OTULIN as represented in Figure 1C and S1B. Protein SQSTM1 and endogenous LC3B were also examined. The numbers are the ratio of LC3B-II/I. (F) OTULIN inhibits LC3B-positive puncta formation. HeLa cells stably expressing mRFP-EGFP-LC3B were transfected with either the vector control or Flag-OTULIN, and treated with H₂O (solvent control) or CQ (20 nM) or CQ (20 nM) + EBSS for 4 h. Cells were fixed and stained with DAPI (blue) and representative fluorescence images of cells treated with H₂O and CQ are shown. Scale bar: 5 μm