Figure 3. The murine ARF autophagy domain maps to amino acids 100 to 120. (A) Western blot analysis of U2OS-ARF cells containing the deletion mutants indicated, untreated or treated with doxycycline (doxy, 0.1 μg/ml) for the indicated time points (hours). Degradation of SQSTM1/p62 (SQSTM1) and accumulation of LC3-II (bottom LC3 band) are indicators of autophagy induction. Actin (ACTB) is included as a loading control. The data depicted are representative of three independent experiments, for multiple clones of each mutant. (B) Western analysis of the level of SQSTM1 and LC3-I and -II following cessation of autophagic flux with 10 mM NH₄Cl. Note that whereas wt ARF induces increased LC3-II, above that which accumulates following NH₄Cl (compare lanes 4 and 3), there is no increase in LC3-II in smARF-induced cells (compare lanes 8 and 7). (C) Electron microscopy of U2OS-ARF cells containing wild-type ARF (wt) or the deletion mutants indicated, untreated (unt) or treated with doxycycline (0.1 µg/mL) for 48 h. The arrowheads point to autophagic vesicles that appear following ARF induction. The average area of autophagosomes, calculated with ImageJ software, is indicated below each panel. The data depicted are representative of three independent experiments, in multiple independent clones of each mutant. Scale bar: 500 nm. (D) Western blot analysis of U2OS-ARF cells containing wild type ARF (wt) or the valine-to-alanine mutant at codon 113 (V113A), untreated or treated with doxycycline (doxy, 0.1 μg/ml) for 24 h. Degradation of SQSTM1 and accumulation of LC3-II are indicators of autophagy induction. Actin (ACTB) is included as a loading control. The data depicted are representative of three independent experiments.