Supplemental Figure 1. GSK induces skin inflammation independent of neuronal TRPC3 in naïve mice. (A) Representative images of Evans Blue
extravasation of mouse hind paws taken 30 min after intraplantar injection of GSK (20 µg; 10 µl in 20% HPCD; right) or vehicle (20% HPCD; 10 µl; left), or Substance P (SP;
50 µM; 10 µl in PBS) or vehicle (PBS; 10 µl; left). (B) Quantification of Evans Blue leakage into the paw induced by GSK, SP or vehicle in naïve mice. n = 5 mice per group. **p < 0.01 vs HPCD or PBS, unpaired Student’s t-test. (C) Strategy for the generation of primary sensory neuron specific Trpc3 knockout mice. Deletion of the
Trpc3 gene in primary sensory neurons was achieved by crossing Trpc3fl/fl mice with PirtCre mice. (D) qRT-PCR analysis revealed a significant reduction in Trpc3 mRNA expression (normalized to Actb) in TG and DRG tissues but not in the thalamus or cerebellum of PirtCre::Trpc3fl/fl mice compared to Trpc3fl/fl controls. n = 5 - 8 mice per group; **p < 0.01 vsTrpc3fl/fl; unpaired Student’s t test. (E) Representative images of Evans Blue extravasation of mouse hind paws taken 30 min after intraplantar injection of GSK (20 µg; 10 µl in 20% HPCD; right) or vehicle (20% HPCD; 10 µl; left) to
PirtCre::Trpc3fl/fl mice and PirtCre negative control littermates. (F) No significant differences in GSK-induced Evans Blue extravasation in the hind paw were observed between PirtCre::Trpc3fl/fl mice and PirtCre negative control littermates. n = 8 mice per group. **p < 0.01 vs HPCD, two-way ANOVA with Bonferroni’s post hoc test.
Table S1. List of DNA primer sequences for quantitative real-time PCR Target gene Forward 5’-3’ Reverse 5’-3’
Trpc3 Il-1β
Il-6 Cxcl10 Cxcl1 Actb
GCGAG AAGTGCTGCGAGA
GGAGAACCAAGCAACGACAAAATA AAAGAG TTGTGCAATGGCAATTCT TGAATCCGGAATCTAAGACCATCAA ATCCAGAGCTTGAAGGTGTTG CTGAATGGCCCAGGTCTGA
TGCACCACCTCGTACTTATGG TGGGGAACTCTGCAGACTCAAAC AAGTGCATCATCGTTGTTCATACA AGGACTAGCCATCCACTGGGTAAAG GTCTGTCTTCTTTCTCCGTTACTT CCCTGGCTGCCTCAACAC
