Figure 4.

Ca²⁺ mobilization induces greater ppERK activation in drug-tolerant than drug-sensitive cells. (A) Immunofluorescence detection of ppERK was performed on drug-sensitive (60 min of BRAFi) and drug-tolerant (3 days of BRAFi) cells in the presence of the Ca²⁺ ionophore ionomycin (iono). The percentage of positive cells and mean ppERK intensity per cell increased in a time-dependent manner after the addition of ionomycin in drug-tolerant cells but not in drug-sensitive cells. (B) Mean ppERK staining intensity per cell (top) and the proportion of cells staining positive for ppERK (bottom) across a time series and concentration range of iono. Note that these graphs are an alternative depiction of the same data shown in (A) but include additional iono concentrations. Iono induced ppERK staining in drug-sensitive cells with concentrations at or above 2.5 µM, whereas ppERK was activated with as little as 39 nM iono in drug-tolerant cells, demonstrating a particular sensitivity to Ca²⁺ mediated activation of ppERK. ppERK staining increased at the earliest time point (5 min) in drug-tolerant cells but not drug-sensitive cells, indicating that drug-tolerant cells respond more rapidly to Ca²⁺. Mean staining intensity in positive cells was significantly elevated at all tested concentrations of ionomycin in drug-tolerant cells but only at the highest concentration in drug-sensitive cells. Moreover, iono activated ppERK in a greater proportion of drug-tolerant cells and did not diminish during the time series at higher concentrations of iono, in contrast to drug-sensitive cells. (C) Representative images of ppERK staining (green) with Hoechst-stained nuclei (blue) under the indicated conditions, each after 15 min of treatment. All images were captured with identical settings and have been set to the same intensity range for visualization. The scale bar shown in the bottom image is applicable to all three images.