Figure 4.

Magnetic activation of MagM8. (a) TRPM8 is mutated (K856A, in orange) and fused to the chimeric ferritin (L^∗H-Ferritin) by a 23 residues linker to create MagM8. (b) Intracellular calcium level in transfected (blue) and NT (black) cells is reported as ΔF/F₀ and shows that MagM8 responds to cooling stimulation (40–15°C). The shaded region and error bars show the mean ± SE based on n = 4 independent cell cultures (slides), containing a total of 1066 transfected cells and 545 NT cells. (c–f) Distribution of intracellular calcium levels over time (as ΔF/F₀) is shown. In the absence of stimulation (c and d), the distribution broadens over time but remains centered near zero. Under periodic magnetic stimulation (275 mT, 0.08 Hz, beginning at t = 30 s), a small percentage of cells (seen in red tail of the distribution) show an increase in calcium-sensitive fluorescence for MagM8 stimulated with a magnet (e) but not for MagM8 exposed to the sham magnet (f). (g and h) Histograms taken from the data in (c)–(f) show the distribution of fluorescence values at 270 s with no magnetic stimulation (black) and with magnetic stimulation (red) (bin size 0.02 ΔF/F₀). These histograms correspond to the white lines in (c)–(f). Vertical red and black lines represent the mean value of these distributions with and without magnetic stimulation, respectively. Error bars show the mean ± SE for each histogram. At 270 s, p = 0.07 for n = slides, p < 0.001 for n = cells for magnetic stimulation; p = 0.53 for n = slides and 0.88 for n = cells for sham stimulation. (i and j) Plotting the mean value of ΔF/F₀ over time shows that MagM8 exposed to a magnetic field displays a statistically significant response to magnetic stimulation, whereas MagM8 exposed to a sham magnet does not. The periodic artifacts in (i) and (j) result from small reversible movement of the microscope stage that sometimes accompanied magnet movement. Shaded regions are mean ± SE calculated with n = independent cell cultures recorded. (k and l) The area under the curve of the calcium activity was calculated for each cell culture. The resulting value for each cell culture is shown as a dot, and the average is shown as a horizonal bar with error bars indicating the corresponding mean ± SE. The significance of the difference between the conditions is indicated as ^∗p < 0.1; ^∗∗p < 0.05; ^∗∗∗p < 0.01. The total number of cells measured from separate cell cultures are (indicated as total number of cells/number of separate cell cultures) with magnet, n = 5184/23 (no stimulation), n = 5372/23 (magnetic stimulation); with sham magnet, n = 2237/18 (no stimulation), n = 2283/18 (sham stimulation). The significance of the increase in calcium activity was assessed using the left-tailed Wilcoxon test. This nonparametric test is used because the increased cell activity is not necessarily normally distributed.