pc-9/GR cells show gefitinib resistance properties, up-regulation of miR-21 and down-regulation of PTEN compared with pc-9 cells.

(A, B) pc-9 and pc-9/GR cells were treated with gefitinib for 72 h in 0.1% serum-containing RPMI. Cell growth was measured by CCK-8 assay in triplicate. Cell growth at 72 h is plotted against gefitinib concentration. (C) gefitinib IC 50 in pc-9 and pc-9/GR cells with triplicate assays performed. **indicates p<0.01. (D) Apoptosis rates of pc-9/GR and pc-9 cells induced by gefitinib were evaluated by flow cytometry. pc-9/GR and pc-9 cells were treated with 1 µmol/L gefitinib for 72 hours before apoptosis evaluation. (E) Relative expression levels of miR-21 in pc-9 and pc-9/GR cells assessed by stem-loop qRT-PCR, and normalized to U6 levels. Triplicate assays were performed for each RNA sample. Significant differences are indicated by *(P<0.05). (F) Pten, p-AKT, AKT, p-ERK, ERK proteins in pc-9 and pc-9/GR cells analyzed by Western blot. (G) Quantitation of western blot analysis showed that Pten protein expression was reduced in PC-9/GR cell compared with PC-9 cells **P<0.01; p-AKT and p-ERK proteins expression were up-regulated in PC-9 GR cells compared with PC-9 cells **P<0.01; there were no significant differences of total AKT and total ERK proteins between PC-9 and PC-9/GR cells.