Figure 4

Confocal visualization of DiI/WS12-containing lipid nanocapsules (LNC600-WS12-DiI) and concurrent LNC-induced [Ca^2±]_i responses in PC3 cell expressing stably TRPM8. (A) Plot (right) compares the dynamics of relative changes in Fluo-4 fluorescence reporting WS12-induced changes in intracellular Ca²⁺ concentration, [Ca²⁺]_i (ΔF/F0, green trace), DiI fluorescence reporting re-distribution of LNC content within the cell (ΔF/F0, red trace) and DiI fluorescence reporting aggregation of LNCs on the cell surface (ΔR/Ro, violet trace). The traces show mean ± S.E.M. signals from 4 cells (image, left). (B) Plots of Fluo-4 (green traces) and intracellular DiI (red traces) responses in 4 cells (numbered in A, left) during the period of interest (POI, brown bar in A, right) highlight the delay (Δt1) between internalization of LNC content and elevation of [Ca²⁺]_i, respectively. (C) The same as B but the delay (Δt2) between the elevation of [Ca²⁺]_i (green traces) and aggregation of LNCs on the cell surface (violet traces) is highlighted. The galleries below the plots show every 8^th (B) and 100^th (C) image captured during the periods corresponding to the plots. (D) Plot displays mean ± S.E.M. values of Δt1 and Δt2 measured in 40 cells upon 17 independent experiments. Note that the LNC is internalized before [Ca²⁺]_i response initiation, while aggregation of LNCs on the cell surface became prominent >3 min later. (E,F) Visualization of the 3-dimensional (3-D) distributions of DiI and Fluo-4 fluorescence 20 min after stimulation of the cell with LNCs. The results are presented by galleries showing (E) every 2^nd x-y image obtained during Z-sectioning protocol (Z-stack) and (F) rotations of the reconstructed 3-D image around Y axis with 30° step (3-D rotations). Note LNC aggregates (red) on the cell surface.