Fig. 3: Differential antifolate toxicity is due to increased drug uptake in the small intestine.

a Representative images of small and large intestine-derived monolayers treated with the indicated methotrexate (MTX) concentration. Nuclei were stained with Hoechst and proliferative cells were stained with EdU. Scale bars, 100 µm. b Quantification of the percent change in proliferative cells relative to untreated cells in small and large intestine-derived monolayers treated with 8 µM MTX and 5 µM pemetrexed (PEM) for 48 h. n = 3 wells. Statistical significance was calculated by a two-way ANOVA followed by Sidak’s multiple comparison test. c Small and large intestine-derived monolayers were incubated with 125 nM tritiated methotrexate (H³-MTX) for 15, 30 or 60 min, then the amount of H³-MTX per well was measured. n = 3 wells. Statistical significance was calculated by an unpaired t-test with Welch’s correction. d Schema for MTX in vivo treatment. e Changes in proliferation in small and large intestines from mice treated with MTX or vehicle. Representative images of small and large intestines stained for Ki67 and Hoechst. Scale bars, 100 µm. Quantification of the average Ki67 intensity per crypt. Boxplot showing median value, whiskers showing lower 10^th and upper 90^th percentiles. n = 180 crypts from 3 mice (small intestine, blue bars) or n = 110 crypts from 3 mice (large intestine, red bars). Statistical significance was calculated by an unpaired t-test with Welch’s correction. f Changes in apoptosis in small and large intestines from mice treated with MTX or vehicle. Representative images of small and large intestines stained for TUNEL and propidium iodide. Scale bars, 100 µm. Quantification of TUNEL⁺ cells per small (blue bars) and large (red bars) intestinal crypts. Boxplot showing median value, whiskers showing lower 1^st and upper 99^th percentiles. n = 60 crypts from 3 mice. Statistical significance was calculated by an unpaired t-test with Welch’s correction. Error bars mean ± SEM. SI: small intestine; LI: large intestine. **** indicates p-values < 0.0001.