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. 2019 Oct 24;20(21):5300. doi: 10.3390/ijms20215300

Figure 4.

Figure 4

USP14 inhibition induces LC3 lipidation. (A) USP14 inhibitor (IU1-47)- and siUSP14-treated A549 cells tested using the annexin-V apoptosis assay. (B) A549 cells were treated with the USP14 inhibitor IU1-47 and then lysed and immunoblotted with anti-USP14, autophagy proteins (LC3 I and II), apoptotic cell death-associated proteins (Bax, Bcl-2, pro-caspase3, and cleaved caspase3), and β-actin antibodies. (C) A549 and H1299 cells were treated with IU1-47 and immunoblotted with anti-USP14 and LC3 I and II antibodies. (D–E) A549 cells were transiently transfected with GFP-LC3B/treated with IU1-47. Fluorescence and Western blotting data were collected for 3 days. Scale bar = 50 μm. (F) A549 cells were treated by USP14 knockdown and immunoblotted with anti-USP14 as well as LC3 I and II antibodies. (G) Lysates from A549 cells were treated with USP14 transfection or IU1-47 were immunoblotted with anti-USP14 beclin-1, LC3 I and II, and β-actin antibodies. (H) A549 cells were treated by USP14 knockdown or beclin-1 knockdown and immunoblotted with anti-USP14 beclin-1, LC3 I and II, and β-actin antibodies.