Eight well chambered coverslips

2D HCT116 and HT29 cells were grown (8 × 10⁴ cells/well) in eight-well chambered coverslips (Ibidi) and treated after 2 days of adherence for 48 h with increasing concentrations of SARB (10, 20, 50 µM). Spheroids (3D) at day 21 derived from SFA and 2D cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at RT, permeabilized with 0.1% TritonX in PBS (15 min at RT), blocked for 30 min with 3% BSA (w/v) in PBS, and stained with a 1:250 dilution of primary antibody against CD133 (Miltenyi Biotec) or 1:100 of ß-Catenin (D10A8) XP (Cell Signaling) overnight at 4 °C. The next day, after a washing step, the spheroids stained with CD133 were incubated with a 1:250 goat anti-mouse dilution of the secondary antibody (Alexa Fluor 555, Invitrogen), where the cells labeled with ß-Catenin were incubated with 1:100 goat anti-rabbit IgG (Alexa Fluor 555, Invitrogen) for 1 h RT, washed again, stained with 1:1000 Hoechst 33342 (Sigma) in PBS (1 mg/ml stock solution) per well and imaged with a Nikon Eclipse Ti–S.

The techniques for real-time fluorescence [Ca²⁺]_m measurements in ASM have been previously described (69 (link), 70 (link)). Briefly, hASM cells plated in eight-well chambered coverslips (Ibidi, Grafelfing, DEU) were incubated in Hanks’ balanced salt solution (HBSS) containing 1 μM Rhod 2-AM (Thermo Fisher Scientific, Waltham, MA) for 45 min at room temperature, medium aspirated, and washed three times in HBSS and incubated further for 15 min to allow for deesterification of the Rhod 2. Cells were then imaged at 1 Hz, 200-ms exposure using Nikon Eclipse Ti imaging system, LED fluorescence light source, and 16-bit high-sensitivity charge-coupled device (CCD) camera with excitation wavelength of 555 nm and emission detected at 580 nm using a 560/55 nm bandpass filter. Baseline fluorescence was established for 30–50 s followed by exposure to nicotine and [Ca²⁺]_m responses monitored in 10–15 cells/chamber using 20 individual, software-defined regions of interest. Inhibitors/activators were added before nicotine exposure (see RESULTS for details).