Fig. 1: Development of an assay for TRPV1 sensitization.
From: Bortezomib-induced neuropathy is in part mediated by the sensitization of TRPV1 channels
a Cells were seeded in a range of 0–6000 cells/well in 384-well plates and tested for calcium flux with KCl and capsaicin. 2000 neurons/well provided robust calcium flux responses and became the default cell density for the assay. b Capsaicin concentration-response curve with an EC50 of 121 nM. c Schematic representation for the assay of TRPV1 long-term sensitization from sensory primary neurons. The potentiation of TRPV1 activation is measured by calculating the AUC after capsaicin administration. d Functional sensitization of TRPV1 channels with a 10-min incubation of known potentiators preceding application of capsaicin (100 nM). All experiments were performed with three biological replicates. *p < 0.5 One-way ANOVA followed Dunnett’s post-hoc test, F6, 14 = 6.468. e Complete, ranked results of responses, each point is an average of two experiments run after 24 h of incubation with test compounds (red) or vehicle (black). % signal change represents overall change in fluorescence intensity normalized to initial intensity levels. f Top 8% of results showing hits that are proteasome inhibitors (Z-Leu3-VS (50 ng/mL), MG-132 (50 ng/mL), and gliotoxin (50 ng/mL)) (arrows). Bars connect two responses measured. Structures of proteasome inhibitors that cause TRPV1 sensitization. Scale bar represents 50 µm.
