Figure 3.

Arf6 regulates autophagosome formation. (A) HeLa cells transfected with two rounds of control or Arf6 siRNA for 5 d were cultured in basal conditions or in starvation medium for 4 h or treated with 100 mM trehalose for the last 20 h and with 400 nM bafilomycin A1 (Baf A1) as indicated for 4 h. Cells were lysed and subjected to Western blotting with the indicated antibodies. The data represent the means ± SD of the percentage of LC3-II/actin ratios obtained from three independent experiments. Black lines indicate that intervening lanes have been spliced out. (B) HeLa cells transfected with two rounds of control or single Arf6 siRNA (n1 to n4) for 5 d were cultured in presence of 400 nM Baf A1 as indicated for 4 h. Cells were lysed and subjected to Western blotting with the indicated antibodies. (C) HeLa cells transfected with two rounds of control or ARNO siRNA for 5 d were cultured in basal conditions or in amino acid and serum starvation medium for 4 h and treated with 400 nM Baf A1 as indicated for 4 h. Cells were lysed and subjected to Western blotting with the indicated antibodies. (B and C) The data represent the means ± SD of the percentage of LC3-II/actin ratios obtained from two independent experiments. SE, short exposure; LE, longer exposure; kd, knockdown.