Fig. 3: Cell proliferation and Lv-cxcl12-MSC-CM.

a Cell viability analysis using PrestoBlue reagent. Irradiated organoids were seeded onto a 96-well plate. Lv-MSC-CM, Lv-cxcl12-MSC-CM and Lv-cxcl12-MSC-CM containing 10 μM of AMD3100 and Lv-cxcl12-MSC-CM containing 10 μM of U0126 were added separately to the wells. Normal organoids served as healthy controls. Each group contained eight wells (n = 8). Six hours later, the organoids were dissociated into single cells. Each 20,000 cells were harvested from each well and stained with 1× PrestoBlue solution for 10 min. Thereafter, the absorbance values were calculated. Data are the mean ± S.D. *P ≤ 0.05: significant increase (Lv-MSC-CM group versus the Normal and IR-alone groups, one-way ANOVA; Lv-cxcl12-MSC-CM group versus AMD3100 and U0126 groups, one-way ANOVA analysis) (Lv-MSC-CM group versus Lv-cxcl12-MSC-CM group). b Activation of Erk1/2 and cell proliferation within irradiated organoids after treatment with Lv-MSC-CM and Lv-cxcl12-MSC-CM for 6 h. Protein levels of Erk1/2, phosphorylated Erk1/2 (pErk1/2) at Thr202/Thr204, PCNA, histone 3 and phosphorylated histone 3 at Ser10 (pHis3 Ser10) were tested by western blotting. β-Actin was used as an internal control. Three independent measurements were performed (n = 3) with similar results. c Phosphorylation of Erk1/2 (pErk1/2) in irradiated crypt cells after treatment with Lv-MSC-CM and Lv-cxcl12-MSC-CM for 6 h. Crypt domains are displayed using white dotted lines. Confocal images were captured. DAPI: nuclei; FITC: pErk1/2. Magnification: 400×; scale bar: 100 μm. e Relationship between CXCL12–CXCR4 and Erk1/2 phosphorylation at Thr202/Thr204. Lv-cxcl12-MSC-CM and Lv-cxcl12-MSC-CM containing 1 μg/ml of CXCL12 neutralizing antibody, 10 μM of AMD3100, or 10 μM of U0126 were used to treat irradiated organoids for 6 h. Thereafter, protein levels of Erk1/2, pErk1/2, PCNA, histone 3 and pHis3 Ser10 were tested by western blotting. β-Actin was used as an internal control. Three independent measurements were performed (n = 3) with similar results. f PCNA expression in irradiated organoid cells 12 h after Lv-MSC-CM and Lv-cxcl12-MSC-CM treatment. Real-time PCR was performed. GAPDH was used as an internal control. The fold-increase was normalized to the Normal group according to the 2^−δδCt algorithm. Data in each group represent the mean ± S.D. of six independent measurements (n = 6). **P ≤ 0.001: significant increase (Lv-cxcl12-MSC-CM group versus IR-alone group, unpaired t test); *P ≤ 0.05 (Lv-cxcl12-MSC-CM group versus Lv-MSC-CM group, unpaired t test). NS: no significance. g PCNA expression in irradiated organoid cells 24 h after Lv-MSC-CM and Lv-cxcl12-MSC-CM treatment. Real-time PCR was performed. GAPDH was used as an internal control. The fold-increase was normalized to the Normal group according to the 2−^δδCt algorithm. Data in each group represent the mean ± S.D. of six independent measurements (n = 6). **P ≤ 0.001: significant increase (Lv-cxcl12-MSC-CM group versus IR-alone group, unpaired t test); NS: no significant difference (Lv-cxcl12-MSC-CM group versus Lv-MSC-CM group, unpaired t test). h β-Catenin stabilization in irradiated organoid cells after treatment with Lv-MSC-CM and Lv-cxcl12-MSC-CM for 6 h. Protein levels of CK-1, GSK3β, phosphorylated GSK3β at Ser9 (pGSK3β Ser9), total β-catenin and active β-catenin were tested by western blotting. β-Actin was used as an internal control. Three independent measurements were performed (n = 3) with similar results