Figure 5.

Ca²⁺ signaling as a sensitive functional endpoint to assess proteasome inhibitor toxicity. The compounds bortezomib (left), carfilzomib (middle), and taxol (right) were investigated regarding their effects on different test endpoints. (A) The PeriTox test was used to assess their effects on neurite area and viability. Horizontal dashed lines at 90% and 75% indicate the cytotoxicity threshold and the neurite effect threshold, respectively. Vertical dashed lines indicate the lowest cytotoxicity-inducing concentration (red) and the concentrations further used for Ca²⁺ imaging experiments (blue). (B,C) Sensory neurons (>DoD38) were pre-treated with the test compounds for 24 h, before Ca²⁺ imaging experiments were performed. (B) The number of cells responsive towards stimulation with the P2X3-specific agonist α,β-methylene ATP was assessed. (C) Baseline fluorescence, indicating the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was quantified for whole sensory neuron cultures. Exemplary single cell fluorescence traces are shown in Figure S9. (A–C) Data are given as % of untreated control cells and are expressed as the mean ± SEM of at least 3 biological replicates. *, p < 0.05; **, p < 0.001; ***, p < 0.0001.