Extended Data Fig. 1: Phylogenetic and functional screening of fungal isolates for IO differentiation-inducing activity.
From: Fungal commensal promotes intestinal repair via its secreted peptide in mice
(a) Experimental workflow for assessing the effect of fungal-spent medium on IO differentiation. Fecal samples were collected from mice and plated on fungal selection media (YPD agar). Individual fungal colonies were isolated and expanded in liquid culture. After inoculation and incubation, the fungal-spent medium was harvested and filtered (0.22-µm filter) for downstream applications. Intestinal crypts were isolated from the mouse intestine and embedded in Matrigel for 3D IO culture. IOs were cultured under proliferation conditions (Noggin, R-spondin1, and Wnt3a), and the filtered fungal-spent medium was added to assess its impact on IO differentiation. (b) Representative images of IOs treated with conditioned media from various fungal isolates. Most isolates fail to induce crypt-like budding, with only isolate #3 (identified as Kp and presented in Fig. 1) displaying differentiation-inducing activity. Non-inducing media from five fungal isolates are displayed as representative images (n = 3). Scale bar, 100 µm. (c, d) Phylogenetic trees constructed using internal transcribed spacer sequences (c) and whole-genome data reveal the taxonomic positioning of Kp relative to other Kp strains (d). (e, f) Representative images (e) and quantification (f; n = 30) of IOs treated with conditioned media from C.albicans (Ca), S.cerevisiae (Sc), or K. pintolopesii (Kp) cultured in DMEM, DMEM + 10% FBS, or SC medium. Versus respective controls, significant differentiation was observed: for Ca (p = 0.35, 0.77, 1); for Sc (p = 0.72, 0.64, 0.37); and for Kp (p = 0.0006, 0.001, 0.0003) across the three media, respectively. Scale bar, 100 µm Data are representative of at least three biologically independent experiments. and are presented as mean ± s.d. Statistical comparisons were conducted using unpaired two-tailed Student’s t-test, *, p < 0.05; **, p < 0.01; ***, p < 0.005; n.s.=no significant change. Panel a created with BioRender.com.
