Fig 1: In vivo validation of compound A using the MWS mouse model.(A–D) Tamoxifen-induced MWS-mediated disease was measured using G-CSF (A), IP-10 (B), IL-6 (C) and SAA (D) in plasma after collection of the blood on day 10. Per group 4 (WT vehicle), 7 (Cre + vehicle) or 8 (Cre + cmpd A) animals were used and mean +/− SEM is depicted. Unpaired two-tailed t-test with welch’s correction in Cre treated vs vehicle samples was performed: **P = 0.0058 (G-CSF), *P = 0.0317 (IP-10), *P = 0.0145 (IL-6), *P = 0.0145 (SAA). (E, F) Levels of compound in plasma in blood collected at day 10, 24 h after last oral dosing. Eight animals were used in this group and mean +/− SEM is depicted. Free compound was calculated (F). (G) Photomicrograph (H&E) of liver sections from a control wildtype (A), vehicle-treated NLRP3 heterozygous (B) and compound A-treated NLRP3 heterozygous (c) mouse. Note the hepatocellular necrosis (yellow arrow), portal vein thrombosis (black arrow), and inflammatory cells infiltration in the sinusoids and portal triads (arrowheads) of the vehicle-treated liver. In contrast, compound A-treated liver appeared histologically comparable to wild-type liver and exhibited only minimal inflammatory cells infiltration (arrowheads) and significant reduction in the severity of thrombosis and necrosis. Bar= 100 microns. (H) Photomicrograph (H&E) of lung sections from a control wildtype (A), vehicle-treated NLRP3 heterozygous (B) and compound A-treated NLRP3 heterozygous (c) mouse. Compound A-treated lung appeared histologically comparable to wild-type liver and exhibited reduction in the severity of thrombosis (arrows) when compared to vehicle-treated NLRP3 heterozygous. Bar = 100 microns.
Fig 2: Loss of TRPV4 reduces tumor growth and cytokine expression known to activate hepatocytes and hepatic stellate cells.(A) Schematic of the orthotopic experiment. (B) Representative images of tumors from WT, Piezo1GFAP KO, or TRPV4-KO mice that were injected with 100,000 KPCY cells in the tail of the pancreas. Organs were collected 20 days after surgery. (C) Quantitative analysis of tumor size. (D–F) Serum levels of IL-6 (D), TIMP1 (E), and SAA (F) were measured by ELISA. (G) Saa2/Saa1 from liver RNA was measured by RT-PCR. Statistical analyses were performed using 1-way ANOVA with Dunnett’s post hoc test. Results are expressed as mean ± SEM.
Fig 3: The TRPV4 antagonist GSK2193874 protects mice from premetastatic niche formation.(A) Graphical illustration of the orthotopic surgery with daily gavage of GSK2193874 (10 mg/kg). (B) Quantitative analysis of tumor weight. (C and D) Serum cytokines levels IL-6 (C) and TIMP1 (D) were measured by ELISA. (E and F) Serum SAA was measured by ELISA (E) and liver expression of Saa1/Saa2 genes (F) by RT-PCR. Statistical analysis was performed using Student’s t test. Results were expressed as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01.
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