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. 2014 Jul 24;9(7):e103305. doi: 10.1371/journal.pone.0103305

Figure 5. Effects of miR-21 knockdown and Pten down-regulation in Pc 9/GR cell lines.

Figure 5

(A) Relative expression levels of miR-21 in pc-9 GR/inhibitor NC+scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were assessed by stem-loop qRT-PCR, and normalized to the levels of U6. Triplicate assays were performed for each RNA sample. Significant differences are indicated by **(P<0.01). (B) Gefitinib sensitivity of pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were evaluated by CCK-8 assay. (C) IC50 values for gefitinib in PC9 GR/21 inhibitor+scramble siRNA(siSCR) transfected cells were significantly lower than in PC9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells(**p<0.01) and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells(#p<0.05). (D) pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were used for cell invasion assays. Invasion cells stained by 0.1% crystal violet were counted 24 h after plating. Upper panels: representative photographs of invasive cells (×200). (E) Number of invasive cells per field was obtained from at least three replicate wells and represented by mean ± SD (bottom graph). **indicates significant difference between pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells (P<0.01). ## indicates significant difference between pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells (P<0.01). (F) Apoptosis rate of pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells induced by gefitinib were evaluated by flow cytometry. All cells were treated with 1 µmol/L gefitinib for 72 hours before apoptosis evaluation. (G) Pten, p-AKT, AKT, p-ERK, and ERK proteins in pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were analyzed by Western blot. (H) Quantification of western blot showed up-regulated expression of Pten protein in PC-9 GR/21 inhibitor+scramble siRNA(siSCR) transfected cells compared with PC-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells (*P<0.05) and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells(#P<0.05); p-AKT and p-ERK proteins expression was down-regulated in PC-9 GR/21 inhibitor+scramble siRNA(siSCR) transfected cells compared with PC-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells (*P<0.05) and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells (#P<0.05). There were no significant differences in total AKT and total ERK proteins among the three cells.