PMC

A–C. Suppression of NNK (10 μM)-induced IGF-1R phosphorylation by blocking Gβγ subunit and PLC but not PKA and Epac in HBE/p53i cells, as determined by Western blot analysis. HBE/p53i cells were pretreated with H-89 (A; 10 μM), ESI-09 (A; 10 μM), gallein (B; 10 μM), and U73122 (C; 1 μM) for 3 h, and then were stimulated with NNK for 15 min. The level of total and phosphorylated IGF-1R was evaluated by Western blot analysis.

A. Immunoblot analysis demonstrating a time-dependent increase in IGF-1R phosphorylation by treatment with NNK (10 μM) in HBE/p53i and BEAS-2B cells. B. Immunoblot analysis demonstrating a dose-dependent increase in IGF-1R phosphorylation by treatment of BEAS-2B cells with NNK (10 μM) for 24 h (n = 3 per group). Densitometric analysis of total and phosphorylated IGF-1R blots (normalized to actin) was performed using the Image J software. Data are presented as the mean ± SD. The statistical significance of difference was determined by Student's t-test (*: P < 0.05). C. Immunoblot analysis demonstrating a time-dependent increase in the phosphorylation of IGF-1R and Akt by treatment with NNK (10 μM) in HBE/p53i cells D. Immunofluorescence staining for the detection of NNK-induced IGF-1R phosphorylation. Cells were stimulated with NNK (10 μM) for 24 h. E. Up-regulation of IGF-1R phosphorylation in HCC-15 cells as determined by fluorescence microscopy.

A and B. Decreases in anchorage-dependent (A; n = 3 per group) and –independent (B; n = 4 per group) colony formation of BEAS-2B cells by treatment with β-AR antagonists. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (*: P < 0.05; **: P < 0.01; ***: P < 0.001). C. Inhibition of NNK-induced foci formation of HBE/p53i cells by treatment with propranolol (n = 3 per group). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (***: P < 0.001). D. Suppression of the NNK-mediated increase in tumor burden by blockade of β-AR (n = 7-14 per group). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (***: P < 0.001). E. Immunohistochemical analysis demonstrating the suppression of IGF-1R phosphorylation and cell proliferation (determined by PCNA expression) in a nodule of the lung from mice treated with NNK and β-AR antagonists. Pro: propranolol; Ate: atenolol; ICI: ICI-118,551.

A and B. Real-time PCR analyses demonstrating IGF2, but not IGF1, IGF1R, and IGFBP3, transcription by stimulation with NNK (10 μM) (A) or isoproterenol (Iso; 10 μM) (B) (n = 3 per group) in BEAS-2B cells. Data are presented as the mean ± SD. Statistical significance of difference was determined by Student's t-test or one-way ANOVA (*: P < 0.05; **: P < 0.01). C. Suppression of NNK-induced IGF2 transcription by treatment with β-AR antagonists in BEAS-2B cells, as analyzed by real-time PCR (n = 3 per group). Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA (*: P < 0.05). Cells were stimulated with NNK (10 μM) in the absence or presence of β-AR antagonists (10 μM) for 24 h. Pro: propranolol; Ate: atenolol; ICI: ICI-118,551. D. NNK-mediated phosphorylation of STAT3 and nuclear translocation of a NF-κB p65 subunit in BEAS-2B cells were analyzed by Western blot analysis. E. Suppression of NNK-induced IGF2 transcription by treatment with Stattic (2 μM) or BAY 11-7082 (BAY; 5 μM) in BEAS-2B cells was analyzed by real-time PCR (n = 3 per group). Cells were stimulated with NNK (10 μM) with or without inhibitors for 24 h. Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA (*: P < 0.05; **: P < 0.01).

A. Immunoblot analysis demonstrating a dose-dependent increase in IGF-1R phosphorylation by treatment with NNK in BEAS-2B cells (n = 3 per group). Densitometric analysis of total and phosphorylated IGF-1R blots (normalized to actin) was performed using the Image J software. Data are presented as the mean ± SD. The statistical significance of difference was determined by Student's t-test (*: P < 0.05; ***: P < 0.001). Iso: isoproterenol. B. Immunoblot analysis demonstrating the activation of IGF-1R by stimulation with β-AR agonists (10 μM) in BEAS-2B cells. Iso: isoproterenol; Dobu: dobutamine; Meta: metaproterenol. C. Inhibition of NNK-induced IGF-1R phosphorylation by treatment with β-AR antagonists (10 μM) in BEAS-2B cells was analyzed by Western blot analysis. Pro: propranolol; Ate: atenolol; ICI: ICI-118,551. Cells were stimulated with NNK for 15 min (left) or 24 h (right) in the presence or absence of indicated inhibitors. In case of a short-term NNK stimulation, cells were pretreated with inhibitors for 3 h. D. Immunoblot analysis evaluating the suppression of IGF-1R phosphorylation by silencing β1- or β2-AR expression in HBE/p53i cells. E. Immunofluorescence staining to detect the modulation of IGF-1R phosphorylation by blockade of β-AR in HBE/p53i cells. Cells were treated with NNK (10 μM) in the absence or presence of atenolol (Ate; 10 μM) or ICI-118,551 (ICI; 10 μM) for 15 min.