Autophagosomes have decreased localization with lysosomal markers in IBMPFD mutant–expressing cells. (A) Epifluorescent images for GFP-LC3 (LC3) and LTR of VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Open arrows highlight autolysosomes (GFP and LTR colocalized), and closed arrows show autophagosomes (GFP only). (B) Pearson's coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments. (C) Epifluorescent images for GFP-LC3 and endogenous Lamp1 immunohistochemistry of U20S, VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells or U20S cells cotreated with Baf transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Arrows highlight autolysosomes (GFP and Lamp1 colocalized). (A and C) The boxed regions in the merge field are enlarged in the adjacent panels. (D) Pearson's coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments. (B and D) Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001. (E) Electron microscopy of tetracycline-inducible control or VCP-WT–, VCP-RH–, or VCP-AE–expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h. Note the accumulation of autophagic structures in the mutant-expressing cell lines. Arrows denote autophagic structures. Bars: (A and C) 15 µm; (E) 1 µm.