Figure 4.

Inhibition of autophagy represses the antiproliferative effect of IBP in lung cancer cells

(A) Cells were transfected with 50 nM siRNA against Atg5 or Beclin1for 48 h and then treated with 10 μg/mL IBP for another 48 h. Cell viability was detected by MTT assay. (B) Cell clone number was counted. (C) Cells were treated with 3-MA or LY294002 in the presence or absence of IBP (10 μg/mL) for 48 h, and then the proliferation rate was measured by MTT assay. (D) A549 and H460 cells were treated with IBP at different concentrations (0, 5, 10, and 20 μg/mL) for 48 h and then lysed and applied to detect phosphorylation of Akt (p-Akt; S473), p-mTOR (S2448), p-p70S6K (S424/T421), and p-4EBP1 (S65/T70) by immunoblotting. Total Akt, mTOR, and 4EBP1 expression was used as the internal control (Ctrl), respectively. (E) A549 cells were transfected with a constitutively active Akt (CA-Akt) for 24 h and were then treated with 10 μg/mL IBP for another 48 h. Cell lysates were collected and performed for immunoblotting of p-Akt and LC3 lipidation. (F) A549 cells were cotransfected with GFP-RFP-LC3 and CA-Akt or vector for 24 h, and then cells were treated with 10 μg/mL IBP for another 48 h. LC3 puncta were detected by CLSM. Scale bar, 10 um. Data are mean ± SD from three independent experiments. One-way ANOVA (Tukey’s post hoc); ∗p < 0.05; ∗∗p < 0.01.