Journal: Oncology Reports

Article Title: RORβ suppresses the stemness of gastric cancer cells by downregulating the activity of the Wnt signaling pathway

doi: 10.3892/or.2021.8131

Figure Lengend Snippet: RORβ exerts anti-proliferative and pro-apoptotic effects on GC cells. (A) MKN45 and AGS cells were transfected with control, RORβ overexpression vector or RORβ shRNA and confirmed by western blotting. The viability of GC cells was evaluated using an CCK-8 assay. Data were analyzed using one-way ANOVA. (B and C) Apoptosis of GC cells was detected using flow cytometry. GC cells were transfected with control or RORβ-overexpression vector for 24 h. Cell apoptosis was evaluated using the values in quadrants 2 and 4. Data were analyzed using a unpaired Student's t-test. (D) GeneChip was used to evaluate the gene expression profiles. The top differentially expressed genes are listed. (E) Protein expression levels of BCL2L11 were analyzed using western blotting. *P<0.05 and **P<0.01. RORβ, retinoic acid-related orphan receptor β; GC, gastric cancer; sh-, short hairpin; BCL2L11, Bcl-2 like protein 11; ov, overexpression.

Article Snippet: GC cells were transfected with 4 μg/μl RORβ-short hairpin (shRNA) and 4 μg/μl control shRNA (both from Santa Cruz Biotechnology, Inc.) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 2 days and then the cells were screened with puromycin (5 μg/ml) for 14 days to obtain the stable RORβ-knockdown cells further verified by western blotting.

Techniques: Transfection, Control, Over Expression, Plasmid Preparation, shRNA, Western Blot, CCK-8 Assay, Flow Cytometry, Gene Expression, Expressing

Journal: Oncology Reports

Article Title: RORβ suppresses the stemness of gastric cancer cells by downregulating the activity of the Wnt signaling pathway

doi: 10.3892/or.2021.8131

Figure Lengend Snippet: 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.

Article Snippet: GC cells were transfected with 4 μg/μl RORβ-short hairpin (shRNA) and 4 μg/μl control shRNA (both from Santa Cruz Biotechnology, Inc.) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 2 days and then the cells were screened with puromycin (5 μg/ml) for 14 days to obtain the stable RORβ-knockdown cells further verified by western blotting.

Techniques: Transfection, Control, Over Expression, Plasmid Preparation, shRNA, Tube Formation Assay, Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction