Fig. 3: RORα/γ inhibit CRC growth and metastasis in vitro and in vivo.

A Representative immunoblots showing the RORα and/or RORγ knockdown and overexpression efficiency in CRC cell lines. B Growth curves of shNC, shRORα and shRORγ CRC cells, as measured with an MTS assay (n = 3). Colony formation assay (C) and transwell assay (D) of shNC, shRORα and shRORγ CRC cells (n = 3). Cells were stained with crystal violet after fixation with methanol. The colonies or migrated cells were counted. Scale bars=200 μm. Representative images (E) and growth curves (F) of subcutaneous tumours derived from shNC and shRORα + γ cells in nude mice (n = 6). Then, the tumours were resected and weighed G. H, I Lung metastasis was evaluated by tail vein injection of shNC and shRORα + γ cells into nude mice. Representative H&E staining of lung tissues is shown (H). The metastatic nodules were counted under a microscope (I). Scale bars=100 μm. J, K Kaplan–Meier estimates of survival times for CRC patients with high or low RORα/γ expression in tumour tissues as evaluated by IHC staining. L Kaplan–Meier estimates of survival times for CRC patients with different RORα/γ expressions. Double positive: high levels of RORα and RORγ. Single positive: high levels of RORα and low levels of RORγ, or low levels of RORα and high levels of RORγ. Double negative: low levels of RORα and RORγ. The cut-off points for RORα or RORγ were determined by the Youden index using receiver operating characteristic analysis. The data are presented as the mean ± SD values. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; Student’s t-test in two groups; One-way ANOVA in more than two groups. ^*P < 0.05, ^**P < 0.01; log-rank test.