Figure S1. Expression of Pkr1 and Prok1 genes in TNBS, acute DSS, and MO-induced colitis. Colitis was induced by intra-colonic instillation of TNBS (A), by supplementation of the drinking water with DSS (B, C), or by intracolonic instillation of mustard oil (D). Modest increases in Prok1 and Pkr1 expression were observed following TNBS-treatment (A), but expression levels were unchanged following DSS- (B, C) or MO- (D) treatment. For the TNBS and MO models, full thickness samples of colon from around the site of chemical instillation were used for the qRT-PCR analyses. Comparable tissue from distal colon was taken from the animals treated with DSS. In each case, values are mean ± SE from n = 6–8 donors per group. Statistical significance in (A) was determined using Student’s t-test. *< 0.05; < 0.01 compared with controls. CT, cycle threshold; Veh, vehicle; D2, day 2; MO, mustard oil.

Figure S2. Inhibition of PROK2-mediated increases in [Ca2+]i by Compound 3. hPKR1 and hPKR2 were transiently transfected using FUGENE6 into the Aequorin parental CHO-A12 cells. Transfected cells were plated and incubated at 37 °C for 18–24 h. For agonist experiments, hPROK2 was added to the cells and luminescence read immediately on a Lumilux Cellular Screening Platform. For antagonist experiments, Compound 3 was added and incubated for 30 min. PROK2 was subsequently added at the EC80 concentration and luminescence read immediately. (A) PROK2 increased [Ca2+]i in hPKR1 and hPKR2 transfected CHO cells. (B) This response was blocked by preincubation of the cells with Compound 3. The data shown are representative traces from a single experiment. Studies were repeated three times to generate the EC50 and IC50 values referred to in the text.

Figure S3. Diclofenac (30 mg kg−1, p.o.) has no effect in models of MO- (A) and TNBS-induced (B) visceral pain. In each case, diclofenac was administered 1 h prior to assessment of visceral allodynia/hypersensitivity. Values shown are mean ± SE from n ≥ 6 donors per group. Data were analyzed using 1-way anova followed by a Dunnett post test with a P value of ≤0.05 being considered statistically significant.

Data S1. Supplementary materials and methods.

NMO_1804_sm_FigS1.tif279KSupporting info item
NMO_1804_sm_FigS2.tif255KSupporting info item
NMO_1804_sm_FigS3.tif91KSupporting info item

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