Whole Gut Transit Time, Fecal Water Content, and Fecal Output

6% (w/v) carmine red (Sigma-Aldrich, St. Louis, MO) with 0.5% methylcellulose (Sigma-Aldrich)

 was dissolved and autoclaved prior to use.

Mice were administered C21 (3 mg/kg) intraperitoneally, and subsequently orally

gavaged with 150 µL of carmine red solution.

Following gavage, mice were single-housed with no bedding for the duration of the experiment, and

animals were not fasted beforehand.

Over the 5 hours following gavage, the time of expulsion was recorded for each fecal

pellet, and each pellet was collected in pre-weighed, 1.5 mL microcentrifuge

tube.

Each pellet collected was checked for the presence of carmine red, and the time of

initial carmine red pellet expulsion was recorded as GI transit time.

The mass of collected fecal pellets was determined, and pellets were left to dry in

an 80 ºC oven for 2 days before weighing the desiccated pellets and calculating

the pellets’ initial water content.

Fecal output rate for each mouse was calculated as the total number of pellets

expelled during the 5 hour time course post-C21 administration divided by the

time the last fecal pellet was expelled.