Figure 3. NNK-mediated IGF-1R phosphorylation is IGF2 dependent.

(a) Secreted growth factors in the conditioned media (CM) from NNK-treated cells were determined by using human growth factor antibody array. (b) BEAS-2B cells were exposed to NNK for the indicated time intervals. Whole-cell lysates (WCL) or conditioned media (CM) were prepared, and NNK-induced IGF1 and IGF2 secretion was determined by Western blot (WB) analysis. Coomassie Brilliant Blue staining (CB) was used as the loading control. (c) Representative images of HBEL/p53i cells stained with IGF2 (green), phalloidin (red) and DAPI (blue) following 24 h culture with or without NNK (left). Graph shows the quantitative analyses of perimembranous IGF2 per field (right; mean±s.d.). Scale bar, 5 μm. (d) Real-time PCR analysis for determining the basal mRNA expression of IGF1, IGF2, IGF1R, and IR in NHBE, HBEL, HBEL/p53i, and BEAS-2B cells. The relative mRNA expression by comparison with the expression level in NHBE cells is depicted (n=3, mean±s.d.). (e) HBEL/p53i cells were pretreated with IGF1- or IGF2-neutralizing antibodies (left) or transfected with IGF2 siRNAs (right). Cells were then exposed to NNK for 15 min. Cell lysates were subjected to WB analysis. (f) Protein lysates from HBEL or HBEL/p53i cells untreated or treated with NNK for 15 min, HBEL cells unstimulated or stimulated with CM from HBEL or HBEL/p53i cells treated with NNK for 15 min (left), and HBEL cells stimulated with CM from NNK-treated HBEL/p53i cells in the presence or absence of IGF2-neutralizing antibody for 15 min (right) were subjected to WB analysis. (g) The protein amounts of IGF1 and IGF2 in CM from HBEL or HBEL/p53i cells were determined by ELISA (n=3, mean±s.d.). (h,i) Modulation of cell viability (n=4, mean±s.d.) (h) and anchorage-dependent colony formation (n=3, mean±s.d.) (i) of BEAS-2B cells stably transfected with control or IGF2 shRNAs and then treated NNK. The statistical significance of differences was determined with the Student's t-test. **P<0.01; ***P<0.001.