Fig 1.
Basal autophagy levels in PC-9/wt and PC-9/gef cells.
(A) Representative Western blot data showed the basal LC3-II levels in PC-9/wt and PC-9/gef cells (B4 and E3). (B) Immunofluorescent staining studies were performed using LC3 antibody to show LC3 puncta in the PC-9/wt and PC-9/gefB4 cells. (C and D): PC-9/wt, PC-9/gefB4 and PC-9/ gefE3 cells were treated with 3-methyladenine (3-MA, 10 mM) and chloroquine (CQ, 1 and 10 μM) for 24 h. Total protein of treated cells was harvested; LC3-I and II levels were analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments.
Fig 2.
Effects of autophagy inhibitors on proliferation of PC-9/wt and PC-9/gef cells.
PC-9/wt, PC-9/gefB4 and PC-9/gefE3 cells were treated with (A) 3-MA (0.1–1 mM) and (B) CQ (5–15 μM) for 96 h. Cell viability was determined using SRB assay. Values are the mean ± S.E.M. (n = 3). *P < 0.05 statistically significant in the 3-MA or CQ-treated groups compared with the controls.
Fig 3.
Effects of gefitinib on LC3-II level in PC-9/wt and PC-9/gef cells.
(A) PC-9/wt, PC-9/gefB4 and PC-9/gefE3 cells were treated with gefitinib (nM) at various concentrations for 24 h. Total protein of treated cells was harvested; LC3-I and II levels were analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments. (B) PC-9/wt and PC-9/gefB4 cells were treated with gefitinib (1 μM) for 24 h. Immunofluorescent staining and fluorescent staining studies were performed using LC3 antibody and LysoTracker red (LysoTR) respectively to show puncta in the cells. Green, FITC-labeled LC3; red, LysoTR; blue, DAPI-labeled nucleus.
Fig 4.
Autophagy activation and cytotoxicity by gefitinib and chloroquine in PC-9/wt and PC-9/gefB4 cells.
(A) PC-9/wt and PC-9/gefB4 were treated with gefitinib (100 nM) and chloroquine (CQ, 5, 10 μM) for 24 h. Total protein of treated cells was harvested. LC3-II levels were analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments. (B) PC-9/wt and (C) PC-9/gefB4 cells were cultured with gefitinib (100 nM) and CQ (5, 10 μM) for 96 h. (D) PC-9/wt cells were cultured with gefitinib (3 nM) and CQ (5, 10 μM) for 96 h. Cell viability was determined using SRB assay. Values are the mean ± S.E.M. (n = 3). *P < 0.05 statistically significant in the gefitinib or CQ-treated groups compared with the controls; # P< 0.05 statistically significant in gefitinib plus CQ-treated groups compared with gefitinib only groups.
Fig 5.
Activation of apoptotic pathway by gefitinib and chloroquine in PC-9/wt and PC-9/gef ells.
PC-9/wt, PC-9/gefB4 and PC-9/gefE3 cells were treated with gefitinib (100 nM) and chloroquine (CQ, 10 μM) for 24 h. Total protein of treated cells was harvested. Procaspase 3, active caspase 3, PARP cleavage levels was analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments.
Fig 6.
The anti-tumor effect of gefitinib and chloroquine in the mouse xenograft model.
Balb/c nude mice bearing PC-9/wt (A) and PC-9/gefB4 (B) xenografts were treated with vehicles as control (○, n = 4 for PC-9/wt and PC-9/gefB4, respectively), gefitinib (50 mg/kg/day by a gavage,◆; n = 5 for PC-9/wt and PC-9/gefB4, respectively), chloroquine (CQ, 75 mg/kg, i.p. ▲; n = 4 for PC-9/wt and PC-9/gefB4, respectively), or a combination of both (■; n = 5 for PC-9/wt and PC-9/gefB4, respectively).Tumors were allowed to grow to 200 mm3 before drug treatments. Values are the mean ± S.E.M. (n = 4–5). ** p<0.001, statistically significant in gefitinib and gefitinib plus CQ groups compared with the vehicle group in PC-9/wt tumor xenografts. * p<0.05, statistically significant in gefitinib plus CQ group compared with the vehicle group; # p<0.05 statistically significant in gefitinib plus CQ group compared with gefitinib alone in PC-9/gefB4 tumor xenografts by Independent-Samples T Test. (C) Representative data show 4 mice with PC-9/wt xenografts and 4 mice with PC-9/gefB4 xenografts which were treated with vehicle, CQ only, gefitinib only and gefitinib plus CQ.