Figure 3.

Disruption of cisplatin-induced autophagic flux by CQ and Baf A1.

Notes: (A) CQ and Baf A1 blocked cisplatin-induced autophagy. Cells were treated with DMSO or 25 µM cisplatin with or without 2 hours pretreatment of either 5 µM CQ or 200 nM Baf A1 for 24 hours. The expression level of LC3-II was detected by Western blot. β-Actin served as internal control. Note that only 1/5 (2 µg) of total protein was subjected to the detection of LC3 level in cells pretreated with Baf A1 to avoid overexposure of the blot. Representative blots from three independent experiments are shown. Quantitative results are shown in the lower panel. Data are presented as fold of control (mean ± SD); *P<0.05 compared to control; ^#P<0.05 compared to cisplatin-treated cells. (B) Increased LC3 puncta in cisplatin-treated cells pretreated with CQ or Baf A1. Data are presented as percentage of green puncta-positive cells from 20 randomly selected images for each condition (mean ± SD); *P<0.05 compared to control; ^#P<0.05 compared to cisplatin-treated cells. (C) CQ interrupted the fusion of autophagosomes to lysosomes in cisplatin-treated cells. Cells were treated with DMSO, 25 µM cisplatin, 5 µM CQ, or 25 µM cisplatin with 2 hours pretreatment of 5 µM CQ for 24 hours. The formation of autophagosomes was detected by transmission electron microscopy. Representative images from three independent experiments with similar results are shown. C1, control cells; C2, cells treated with 25 µM cisplatin; C3, cells treated with 5 µM CQ; and C4, cells pretreated with 5 µM CQ and 25 µM cisplatin. C4-1 and C4-2, magnified view of C4. Scale bar =2 µm in C1–C4; 1 µm in C4-1 and C4-2. Black arrows indicate autophagosomes or autophagolysosomes. Note that autophagosomes were located beneath lysosomes in C4-1 and C4-2, indicated by *.

Abbreviations: SD, standard deviation; DMSO, dimethyl sulfoxide; LC3, light chain 3; L, lysosomes; N, nucleus; Baf A1, bafilomycin A1; CQ, chloroquine.