Enteroid Luminal DON Exposure Protocols

For basolateral DON exposure, DON (final conc. 1 µM; Sigma–Aldrich Co. LLC, St. Louis, MO, USA) was added to the enteroid culture medium. In contrast, luminal DON exposure was conducted through DON microinjection (28.3 µM in DPBS) into enteroids. Enteroid lumen microinjection and volume measurement of enteroid lumen and microinjection were done as previously described [20 (link)]. The final concentration was equivalent to 1 µM in microinjected enteroids (Table S1). Briefly, the tip of the needle (Femtotips II; Eppendorf AG, Hamburg, Germany) was broken off and its inner diameter was adjusted to 3–4 µm. The 28.3-µM DON solution (final conc. 1 µM) was loaded into the needle using a microloader tip (Eppendorf AG, Hamburg, Germany). Microinjection was performed using a microinjector (FemtoJet^® 4i; Eppendorf AG) and manipulators (MM-92 and MMO-202ND; Narishige Scientific Instrument Lab, Tokyo, Japan) under a microscope (Olympus Co., Tokyo, Japan). Enteroid lumen was stained with 1 µM rhodamine123 (Rh123) for 3 h (Figure S1B,C) to measure its volume. Likewise, DPBS was microinjected into the enteroids to test the effects of microinjection itself (described as “PBS injection” in the figures). For the experiment to test dextran permeability, DPBS was microinjected into the enteroids in the basolateral DON exposure group before exposure in view of the effects of microinjection itself.