Fig 2: Stromal cells serve as a source of WNT ligands upon exposure to 12 Gy TBI. (A) RT-qPCR Profiler PCR array screening of WNT signaling pathway–related genes in stromal cells isolated from Bmi1-CreER mice exposed to 12 Gy TBI vs sham-treated mice at 24-hour time point. (B–E) RT-qPCR analysis of WNT ligand encoding genes’ expression: (B) Wnt2b, (C) Wnt4, (D) Wnt5a, and (E) Rspo3 in stromal cells isolated from Bmi1-CreER mice exposed to 12 Gy TBI vs sham-treated mice at 24 hours. Data points represent the average of 5 independent experiments with the mean ± SD indicated. Significance was determined by the Student’s test followed by an analysis of the normal distribution (Tukey’s test). ***P < .001.

Fig 3: Stromal cell–derived WNT ligands stimulate regeneration of Bmi1-CreERderived organoids. (A) Representative merged (bright field and GFP channel) images of Bmi1YFP–derived organoid regeneration on day 8, cultured in L-WRN conditioned medium or ADF medium with or without stromal cells obtained from nonirradiated or irradiated source with or without 10 µM LGK974. Scale bar = 50 µm. (B) Quantification of the area of regenerating organoids. Data points represent the average of 5 independent experiments with the mean ± SD indicated. (C, D) ELISA analysis of the (C) WNT5A and (D) RSPO3 levels secreted by stromal cells isolated from Bmi1-CreER mice exposed to 12 Gy TBI vs sham-treated mice at 24 hours. Data points represent the average of 3 independent experiments with the mean ± SD indicated. Significance was determined by 1-way analysis of variance for panel B and the Student’s test for panels C and D followed by an analysis of the normal distribution (Tukey’s test). *P < .05; **P < .01; ***P < .001; ****P < .0001.