(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. 