Tunneling in NCL-SG3 cells. (A) Localization of ANO1 by immunocytochemistry and mCh-STIM1 before (Ctr) and after store depletion (Tg). An intensity plot performed along a line (arrow) crossing a STIM1 cluster illustrates the separation of STIM1 and ANO1 at the PM focal plane. (B–D) Comparative localization at the PM plane of mCh-STIM1 with ANO1–GFP and the endogenous ANO1 protein (ANO1–Ig) following store depletion. As indicated by the PCC and the peak-to-peak distances the two proteins do not colocalize laterally but are localized at the same optical plane (z distance) (n = 8–18; one-way ANOVA, P < 0.0001 for B and C, P = 0.7 for D). (E) Intracellular Ca²⁺ elevation induced by trypsin application in NCL-SG3 cells loaded with Fluo4-AM. The application of the SOCE inhibitor BTP-2 (10 μM) reduces the amplitude and the duration of Ca²⁺ release (n = 379/402; unpaired t test, P < 0.0001). (F) Confocal images of NCL-SG3 cells expressing the Cl⁻ sensor mbYFPQS and mCh-STIM1 after store depletion. The orthogonal section through the cell indicates the PM localization of the chloride sensor. (G) Kymographs were measured during a tunneling event on cells expressing mbYFPQS and mCh-STIM1. The line passes through a SOCE cluster and an adjacent cell appendage labeled by mbYFPQS. The changes in Cl⁻ concentration (upper) are distal from the STIM1 cluster, which indicates the Ca²⁺ entry point. (H) Time course of the amplitude of the Cl⁻ signal induced by Ca²⁺ tunneling from NCL-SG3 cells in the same dish that either do not show any CaTAr2 expression (Ctr) and cells expressing CaTAr2. (I) Violin plots summarizing the amplitude of the Cl⁻ signal 3 min after trypsin and Ca²⁺ addition to stimulate tunneling (n = 32–33; unpaired t test, P = 0.0004).