Figure 5: NNK-mediated increase in intracellular Ca²⁺ is important for IGF2 secretion and IGF-1R activation.

(a) BEAS-2B/GFP-IGF2 cells were stimulated with NNK for 15 min after pretreatment with the mecamylamine (MCA) for 3 h. IGF-1R phosphorylation in WCL or IGF2 secretion in the CM were determined by western blot (WB) analysis. (b,f) BEAS-2B/GFP-IGF2 cells were transfected with indicated siRNA, and then stimulated with NNK for 15 min. IGF-1R phosphorylation in WCL or IGF2 secretion in the CM were subjected to WB analysis. (c,d) HBEL/p53i cells were treated with ionomycin (Iono) or thapsigargin (Thapsi) for 15 min (c) or incubated with indicated concentration of Ca²⁺ and NNK for 15 min (d). Activation of IGF-1R or Akt was determined by WB analysis. (e,g) HBEL/p53i or BEAS-2B/GFP-IGF2 cells were stimulated with NNK for 15 min after pretreatment with the indicated inhibitors for 3 h. IGF-1R phosphorylation in WCL or IGF2 secretion in CM were subjected to WB analysis. BAP: BAPTA-AM; Amlo: amlodipine; Nife: nifedipine. (h) Time-lapse imaging analysis for GFP-IGF2 secretion from BEAS-2B/GFP-IGF2 cells. Cells were pretreated with amlodipine (Amlo) or nifedipine (Nife) for 3 h and further stimulated with NNK. White arrows indicate secreted GFP-IGF2. Right: secreted GFP-IGF2 out of 25 BEAS-2B cells stimulated with NNK in the presence or absence of indicated inhibitors at 30 min after NNK treatment was quantified using Harmony high content imaging and analysis software. Data are presented as the mean±s.d. ***P<0.001, determined by Student’s t-test. Scale bar, 20 μm. (i) Time-lapse imaging of Fluo-4 AM fluorescence signals from HBEL/p53i cells. Cells were pretreated with amlodipine (Amlo; 1 μM) or nifedipine (Nife; 1 μM) for 3 h, and were exposed to NNK (10 μM) for 30 min. The changes in fluorescence intensity were plotted to show the change in intracellular Ca²⁺ levels. Data were normalized to the average fluorescence intensity measured before NNK treatment in the HBEL/p53i cells (n=4, mean±s.d.).