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. Author manuscript; available in PMC: 2012 Jun 13.
Published in final edited form as: Int J Cancer. 2011 Aug 3;130(8):1733–1744. doi: 10.1002/ijc.26164

Figure 2.

Figure 2

(a) Stable knockdown of mutant KRAS expression by retroviral-mediated shRNA in H1792 and H2122 cells, both of which harbor the KRAS G12C mutation (GGT to TGT) in codon 12. P: parental cells, C: pRS (non-targeting shRNA) vector-infected cells, K: pRS-KRAS-C12-infected cells. Thirty micrograms of whole cell lysate was loaded per lane, and western blots were performed using α-Tubulin expression levels as a loading control. (b) shRNA-mediated KRAS knockdown reduces IL-8 mRNA expression in H1792 and H2122 cells as determined by quantitative real-time RT-PCR. *p < 0.01 for comparison between KRAS knockdown cells and the parental cells by Kruskal–Wallis test with Dunn’s multiple comparisons. Mean levels of the parental cells are set at 100%. (c) shRNA-mediated KRAS knockdown reduced IL-8 protein levels in cultured medium of H1792 and H2122 cells as determined by ELISA assay. *p < 0.001 for comparisons between KRAS knockdown cells and the parental cells by ANOVA with Bonferroni multiple comparisons. Column represents mean ± SD from four independent experiments. (d) Treatment with gefitinib or erlotinib transcription ally down-regulated IL-8 expression in HCC827 NSCLC cells harboring EGFR mutations. After treatment with gefitinib (1 μM) or erlotinib (1 μM) for 24 hr, cells were harvested for quantitative real-time RT-PCR analysis. Columns represent means ± SD from four independent experiments. *p < 0.001 for comparison of Mock treatment (DMSO alone) by ANOVA with Bonferroni multiple comparisons. (e) Treatment with gefitinib or erlotinib reduced IL-8 protein levels in cultured medium of HCC827 cells. After treatment with gefitinib (1 μM) or erlotinib (1 μM) for 12 or 24 hr, cultured medium was collected, and IL-8 concentration was determined by ELISA assay. Columns represent means ± SD from three independent experiments. *p < 0.001 for comparison of Mock treatment (DMSO alone) by ANOVA with Bonferroni multiple comparisons. (f) siRNA-mediated EGFR knockdown in HCC827 cells as evaluated by quantitative RT-PCR. NT: treatment with medium alone; Mock: treatment with Tax siRNA. Two siRNAs targeting different sites of EGFR mRNA (EGFR-1 and EGFR-2) were used. Columns represent the mean ± SD from four independent experiments. *p < 0.0001 for comparisons between NT and each siRNA treatment by ANOVA with Bonferroni multiple comparisons. shRNA-mediated EGFR knockdown reduced IL-8 expression at (g) mRNA and (h) protein levels in HCC827 cells as evaluated by quantitative real-time RT-PCR and ELISA assays. Twenty-four after siRNA transfection, the medium was replaced by fresh medium. After 48 hr, cells and supernatants were harvested for the assays. *p < 0.0001 for comparisons between NT and each siRNA treatment by ANOVA with Bonferroni multiple comparisons. Mean levels of NT are set at 100% for Fig. 2F and 2G.