Fig. 5
From: Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting RORβ
RORβ inhibits tumorigenesis and the self-renewal of CCICs. a RORB expression in colorectal cancer cells. RORB mRNA and protein expression levels were detected in colorectal cancer cells by Taqman RT-qPCR and western blotting, respectively. RORB mRNA was normalized with GAPDH. b RORβ expression in primary colorectal cancer tissues. RORβ expression in human primary colorectal cancer tissues was detected by IHC staining with antibodies against RORβ. Normal rabbit IgG was used as a negative control. c RORB mRNA in primary colorectal cancer tissues. RORB mRNA was measured by Taqman RT-qPCR in 14 patients with colorectal cancer. The results showed that the levels of RORB in colorectal cancer cells were significantly lower than those in matched adjacent tissues. *p < 0.05 (ANOVA). d Tumorigenicity of RORβ-overexpressing cells. RORβ- overexpressing SW620 cells (1 × 106) as well as their control cells infected with blank lentivirus were injected into naked Balb/c mice, respectively (n = 5). Tumor formation was quantified within 4 weeks. *p < 0.05 (ANOVA). The results showed that RORβ inhibited tumor growth. e Quantification of colospheres in RORβ-overexpressing cells. Colospheres were counted in RORβ-overexpressing P1 and control cells infected with blank lentivirus at the 5th day under low-adhesion and serum-free condition. The number of colospheres significantly decreased after RORβ overexpression compared with the controls. *p < 0.05 (ANOVA). f Determination of the percentage of CD44 + CD24+ cells after overexpression of RORβ. The percentage of CD44 + CD24+ cells were analyzed by FCM in RORβ-overexpressing HT29, P1 and SW620 cells, with cells infected with blank lentivirus as controls. The results showed that RORβ reduced the percentage of CD44 + CD24+ cells compared with the control cells, all p < 0.05 (ANOVA). g RORβ expression in RORB-knockdown cells. Cells were infected with lentivirus encoding RORB shRNA for 72 h and subsequently screened with 5 μg/mL Puromycin for 7 days. The surviving cell clone was picked out with limiting dilution analyses. RORβ was detected by western blotting in these RORB-knockdown clones, with cells infected with scrambled shRNA lentivirus as controls. h Quantification of colospheres in RORB-knockdown cells. Colospheres from the above RORB-knockdown colorectal cancer cell clones were counted at day 5 under serum-free conditions. The number of colospheres was significantly higher in RORB-knockdown cells than in the control cells. *p < 0.05 (ANOVA)