Fig. 2: Unbiased screen uncovers proteasome bortezomib as a long-term TRPV1 sensitizer.

a Bortezomib concentration-dependent increase in TRPV1 sensitization. b Left, representative images of calcium flux in sensory neurons in response to capsaicin 30 nM (CAPS) after treatment with SES buffer (top) or after 24 h pre-incubation of bortezomib 100 nM (bottom). Right, individual fura-2 based calcium tracings of DRG neurons after a 24 h treatment with a single application of SES buffer (top) or treated with 100 nM bortezomib for 24 h (bottom). c Average tracing for all TRPV1+ cells after incubation of SES buffer or bortezomib, includes a low dose of capsaicin (30 nM) followed by a higher dose of capsaicin (1 µM) to activate any remaining capsaicin-sensitive neurons in vitro, and then finally a high dose of KCl (40 mM) to define the dish population of responsive neurons. Three replicates are shown per condition. d Proportion of DRG neurons responsive after exposure to 30 nM capsaicin after bortezomib treatment. Mean and standard error from three different trials. Dashed line represents maximum proportion of TRPV1+ neurons responsiveness. *: p < 0.5 Two-way ANOVA followed by Sidak’s post-hoc test. F^{10, 40} = 4.751. e Quantile-quantile analysis of the number of neurons with TRPV1 sensitization after various times of pre-incubation of bortezomib. The X-axis represents the unitless area under the curve (AUC) response of control cells to a first dose of 30 nM capsaicin compared with the AUC of an interpolated matching quantile experimental cell exposed to a first dose of 30 nM capsaicin following treatment with 100 nM bortezomib for the times indicated. The dotted line with a slope of 1 indicates the null-hypothesis, where the two populations of cells behave similarly upon activation by capsaicin. Bending towards the Y-axis indicates stronger responses to capsaicin among cells pre-treated with bortezomib. Scale bar represents 40 µm.