Figure S1 Pyr3 acutely blocks a reconstituted CRAC pore in a dose-dependent, non-reversible manner as well as affecting sodium influx through it. (A) Time course of measured currents (n = 4 cells) of acute Pyr3 inhibition of CRAC in a HEK293 cell system expressing STIM1 and Orai1 to reconstitute the CRAC pore. (B) Time course of acute Pyr3 inhibition (3μM) in native RBL-2H3 cells (n = 6 cells). (C) Time course of currents (n = 8 cells) with acute Pyr3 inhibition of a reconstituted CRAC pore. Pyrazole compound was applied for only a limited time to examine reversibility of inhibitory effect. (D) Time course of Pyr3-effect (10 μM) on divalent-free sodium current through CRAC-pore after activation of current in Ca2+ containing buffer (n = 4 cells). All values are mean values ± SEM.

Figure S2 Pyr10 potently blocks DAG-mediated TRPC3 membrane currents. Mean values of OAG-induced (100 μM) TRPC3 currents in HEK293 cells in the absence and presence of 3 μM Pyr10. Mean values ± SEM of (n = 6).

Figure S3 Selectivity of Pyr10 and Pyr6 on TRPCs. Average inhibition of peak calcium entry level measured by Fura 2 Ca2+ imaging at 10 μM Pyr6 or Pyr10 in HEK293 cells overexpressing TRPC4beta-YFP, YFP-TRPC5 or GFP-TRPC6. HEK293 cells were challenged with carbachol (100 μM) to stimulate calcium entry and pre-incubated for 5 min with corresponding pyrazole compounds. Values are presented as percentage of non-inhibited carbachol-stimulated cells from the same experiments and were derived from a total of 27–55 cells from three individual experiments.

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