RORβ inhibits stemness properties of GCSCs. GC cells were transfected with control, RORβ overexpression vector or RORβ shRNA vector. (A) Images of colospheres of GC cells. Self-renewal capacity of GCSCs was determined using a sphere formation assay. (B and C) mRNA expression levels of CSC markers in RORβ-overexpressing or RORβ-silenced AGS cells and MKN45 cells were analyzed using RT-qPCR and confirmed by western blotting. (D and E) Tumorigenicity of RORβ-overexpressing cells. Nude mice were inoculated with 1×105, 5×105 and 1×106 RORβ-overexpressing AGS cells (n=5). The numbers and volumes of the tumors were observed within 4 weeks. (F) RNA and proteins from the tumors were extracted. Expression levels of EMT markers in the aforementioned tumors were analyzed using RT-qPCR and western blotting. (G) RNA and proteins from the tumors were extracted. Expression levels of GCSC markers in the aforementioned tumors were analyzed using RT-qPCR and western blotting. Data were analyzed using a unpaired Student's t-test. *P<0.05 and **P<0.01. RORβ, retinoic acid-related orphan receptor β; GCSCs, GC stem cells; GC, gastric cancer; CSCs, cancer stem cells; RT-qPCR, reverse transcription-quantitative PCR; ov, overexpression; sh-, short hairpin.