Functional VCP is required for autophagosome maturation. (A) Epifluorescent images for GFP and mRFP in U20S or VCP-WT–, VCP-RH–, VCP-AE–, or VCP-EQ–expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD– or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001. (D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)–expressing cells. Bars, 15 µm.