Paneth cells acquire multi-potency upon Notch activation after irradiation

In murine models, we find that irradiation of Paneth cells caused a gain of a stem cell-like transcriptome and induced activation of the Notch signaling pathway. This study documents plasticity by Paneth cells, a fully committed cell population to participate in epithelial replenishment following stem cell loss. Single-cell dissociation was carried out as previously described (Li et al., 2016; Sato et al., 2011). Cell pellets were washed with cold PBS and re-suspended in FACS buffer. Cells were stained with DAPI, PerCP/Cy5.5-conjugated EpCAM, BUV395-conjugated CD45, and APC/fire 750-conjugated CD24. Cell suspensions were subjected to sorting by BD Biosciences Aria II Flow Cytometer. Single viable intestinal epithelial cells were gated by forward scatter, side scatter, and by negative staining for DAPI and CD45, and positive staining for EpCAM. Subpopulations were further gated based on CD24 and tdTomato (using R-phycoerythrin/PE channel). Paneth cells (tdT+CD24+) and derivative (tdT+CD24-) cells were FACS-sorted from irradiated (5 days after radiation) and non-irradiated 8-14 week old Lyz1CreER; R26R-tdT mice with one dose of tamoxifen adminstration (10mg/mouse), and subjected to total RNA extraction using Qiagen RNeasy Plus Micro kit.