Inhibition of autophagy enhanced the toxicity of 3,4,4′-THS in A549 cells. (A) A549 cells were treated with 3,4,4′-THS (40 μmol/L), 3-MA (5 mmol/L), <t>wortmannin</t> (2 μmol/L) or a combined treatment of 3,4,4′-THS and 3-MA or wortmannin for 3, 6, or 12 h. Microscopic photographs (400×) were taken under an inverted phase contrast microscope. (B) Cell viability determined by MTT assay. (C) Histogram shows the ratio of TUNEL-positive cells. (D) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. 3-MA was added to the cells for 1 h prior to 3,4,4′-THS treatment. (E) Histogram shows the relative level of cleaved PARP. (F) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. Wortmannin was added to the cells for 1 h prior to 3,4,4′-THS treatment. (G) Histogram shows the relative level of cleaved PARP. Data are expressed as the mean (±SEM) of three independent experiments. b P