Fluo 4

Cells (1×10⁵ cells/ml), including untransfected and shRNA-transfected H1299 cells, were seeded onto glass chamber slides (043320B; Shengyou Biotechnology, Hangzhou, China). Cells were cultured for 24 h, and the medium was then removed and replaced with Hanks balanced salt solution (HBSS) containing 1 µM Fluo-4 (cat no. F312; Dojindo Laboratories Inc.), after which the cells were incubated for 1 h at 37°C. Cells were then washed once with HBSS and stored in fresh HBSS for 30 min prior to further experimentation. Nicotine was directly added into the chamber at a final concentration of 1 mM. In some chambers, the cells were pre-incubated with α-BTX at a final concentration of 1 µM for 30 min at 37°C prior to the addition of nicotine. Immediately after the addition of nicotine, Fluo-4 was excited with an Argon laser (the excitation wavelength was 494 nm and emission wavelength was 519 nm) using a Zeiss LSM-710 EXCITER microscope (Zeiss, Thornwood, NY, USA) to assess changes in the calcium flux from the nAChR ion channels. The fluorescence intensity of calcium was recorded as the ratio of F and F0, where F represented the peak fluorescence intensity of the cellular calcium influx when stimulated by nicotine, and F0 represented the basic fluorescence intensity of cellular calcium influx. The mean relative fluorescence of the peak was calculated as [(F-F0)/F0] × 100%

Intracellular calcium concentration ([Ca²⁺]i) in INS‐1 cells was measured with a Calcium kit Fluo‐4 (#CS22; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). INS‐1 cells were placed on a φ35‐mm glass bottom culture dish. Growth medium was replaced with loading buffer containing 3 mmol/L glucose and Fluo4‐AM, and the culture dish was incubated at 37°C for 1 h. Loading buffer was then replaced with recording buffer containing 3 mmol/L glucose. The Fluo‐4 fluorescent signal over the INS‐1 cell area was detected through excitation at 488 nm using an LSM 880 Live microscope (Carl Zeiss, Oberkochen, Germany).