Fig 1: Rh2 enhances the therapeutic efficacy of radiation in CT26/luc tumor-bearing mice. (A) Flow chart of in vivo experiments. CT26/luc cells were subcutaneously implanted into the right flanks of the male mice. Mice were randomly divided into four groups (n = 6 per group): control, 4 Gy irradiation, Rh2 alone (10 mg/kg, gavage), and a combination of both (pretreated with 10 mg/kg Rh2 one day prior to 4 Gy irradiation). (B) Tumor volume was significantly inhibited in all treatment groups, especially in the combination group. (C) The expressions of tumor progression-related proteins, such as MMP-9, VEGF, Bcl-2, and Cyclin D1, were all increased in the radiation-alone group but were decreased in Rh2-alone and combination groups. The cleaved caspase-3 and -8 were increased in all treatment groups. Phosphorylated MAPK proteins, such as Akt, Erk, JNK, and p38, were all increased in the radiation-alone group but decreased in Rh2-alone and combination groups. (D) The splenocytes of the spleens were used to assay the expressions of CTLA-4 and PD-1. Both CTLA-4 and PD-1 could be slightly reduced by radiation and Rh2-alone treatments. Notably, PD-1 but not CTLA-4 was significantly reduced by the combination treatment. * p < 0.05, ** p < 0.01, and *** p < 0.001 for the treatment groups compared with that of the control. ## p <0.01 and ### p< 0.001 for Rh2-alone and combination groups compared with that of the radiation-alone group. $$ p < 0.01 and $$$ p < 0.001 for radiation-alone and combination groups compared with that of the Rh2-alone group.