Fig. 3

BCN057 rescued Lgr5⁺ ISC and induced a regenerative response in vivo. a (i) Time-course study on the effect of abdominal irradiation (AIR) on Lgr5-positive ISC. Representative images of jejunal sections demonstrating the presence of green fluorescent protein (GFP)⁺Lgr5⁺ ISC (indicated with arrow) in Lgr5/GFP-IRES-Cre-ERT2 knock-in mice up to 24 h post-AIR. All the ISC at the crypt base disappeared at 72 h post-AIR. (ii) The number of Lgr5⁺GFP⁺ ISC per crypt in jejunal sections from Lgr5/GFP-IRES-Cre-ERT2 knock-in mice at different time points post-AIR. The number of Lgr5⁺GFP⁺ ISC per crypt reduced at 24 h post-irradiation (*p < 0.04). At 72 h post-AIR, most of the Lgr5⁺GFP⁺ ISC disappeared (p < 0.0001). b (i) Representative images of jejunal sections at 3.5 days post-AIR demonstrating the presence of GFP⁺Lgr5⁺ ISC (indicated with arrow) in Lgr5/GFP-IRES-Cre-ERT2 knock-in mice receiving BCN057 at 24 h post-AIR. Note the absence of GFP⁺ cells in mice receiving only AIR. (ii) The number of Lgr5⁺GFP⁺ ISC per crypt in jejunal sections from Lgr5/GFP-IRES-Cre-ERT2 knock-in mice exposed to irradiation and then treated with BCN057. The number of Lgr5⁺ cells are significantly higher in BCN057-treated irradiated mice compared with AIR controls (*p < 0.0001). Unirradiated mice receiving BCN057 also demonstrated a higher number of Lgr5⁺ cells at the crypt base compared with AIR controls (*p < 0.0002; unpaired t test, two-tailed). (iii) Representative images of jejunal sections demonstrating the presence of Ki67 in Lgr5⁺GFP⁺ ISC localized in the crypt base. Representative images from the single fluorescence channel showed localization of Lgr5⁺GFP⁺ cells (green, indicated with yellow arrow head) and Ki67⁺ cells (red, indicated with green arrow head). Cells that are double-positive for Ki67 and GFP are indicated with white arrows in both the single fluorescence channel and in the merged image. (iv) The percentage of Lgr5⁺GFP⁺/Ki67⁺ in jejunal sections from Lgr5/GFP-IRES-Cre-ERT2 knock-in mice exposed to irradiation and then treated with BCN057. The percentage of Lgr5⁺GFP⁺/Ki67⁺ cells are significantly higher in BCN057-treated irradiated mice compared with AIR controls (*p < 0.0001). Unirradiated mice receiving BCN057 also demonstrated a higher percentage of Lgr5⁺GFP⁺/Ki67⁺ cells at the crypt base compared with AIR controls (*p < 0.0003; unpaired t test, two-tailed). c (i) Representative image of jejunal sections at 48 h post-AIR demonstrating the presence of TUNEL-positive apoptotic cells at the crypt base in mice exposed to AIR. However, mice receiving the BCN057 treatment at 24 h post-AIR did not show any TUNEL-positive cells. (ii) Percentage of TUNEL-positive apoptotic cells in jejunal sections from mice exposed to AIR. The percentage of TUNEL-positive cells are significantly higher in the AIR group compared with mice receiving BCN057 at 24 h post-AIR (p < 0.0002). d (i) Schematic representation of the treatment schema for lineage tracing assay in Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice. (ii) Confocal microscopic images (×40) of the jejunal section from Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice. tdTomato (tdT)-positive cells are shown in red; Lgr5⁺GFP⁺ cells are shown in green. Nuclei are stained with DAPI (blue). Marked expansion of tdT-positive red cells above the +4 position (representing transit amplifying cells) were noted with BCN057 treatment. Please note the presence of yellow cells at the bottom of the crypt representing tdT-positive and GFP⁺Lgr5⁺ ISC (yellow due to red + green). (iii) Confocal microscopic images (×10) of the jejunal section from Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice. Please note the presence of villi containing red tdT-positive cells (regenerative villi) in unirradiated controls or BCN057-treated mice. In the absence of BCN057 treatment, no tdT-positive cells were noted in irradiated mice jejunum. (iv) The number of regenerative villi. Irradiated mice receiving BCN057 showed a significantly higher number of regenerative villi compared with irradiated controls (*p < 0.002). Un-irradiated mice receiving BCN057 also demonstrated a higher number of regenerative villi compared with irradiated controls (*p < 0.0006)