(D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A-C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm. 