Figure 4. Simultaneous recording of free ER Zn²⁺ and cytosolic free Ca²⁺ in response to CPA, thapsigargin or ATP treatment of MIN6 cells.

MIN6 cells expressing eCALWY-4 (a,c,e) or ER-eCALWY-4 (b,d,f) were incubated with Fura-2 (3 µM) in KHB for 20 minutes before acquisition. Fluorescence intensity ratios were determined sequentially for eCALWY-4 sensors and Fura-2. After 3 minutes of perfusion with KHB, cells were treated with different drugs. Acquisition rates were 20 images per minutes for CPA and thapsigargin experiments, and 30 images per minute for ATP experiments. Results displayed are representatives of the responses obtained. (a) MIN6 cells expressing eCALWY-4 were treated with 20 µM CPA (n = 6 cells) (b) MIN6 cells expressing ER-eCALWY-4 were treated with 20 µM CPA (n = 6 cells) (c) MIN6 cells expressing eCALWY-4 were treated with 1 µM thapsigargin (n = 9 cells) (d) MIN6 cells expressing ER-eCALWY-4 were treated with 1 µM thapsigargin (n = 4 cells) (e) MIN6 cells expressing eCALWY-4 were treated with 100 µM ATP (n = 9 cells) (f) MIN6 cells expressing ER-eCALWY-4 were treated with 100 µM ATP (n = 3 cells). Data are means ± S.E.M.

(g,h) Mean ratios were calculated before (1) and during (2) calcium response for represented traces and for an acquisition time of 1 minute. (g) Mean ratios upon CPA, thapsigargin and ATP treatment for MIN6 cells expressing eCALWY-4. (h) Mean ratios upon CPA thapsigargin and ATP treatment for MIN6 cells expressing ER-eCALWY-4. Error bars represent S.D.