Fig. 2

NRIP2 is significantly upregulated in CCICs. a Screening strategy for NRIP2. NRIP2 was identified in a screen for colosphere formation using a retroviral cDNA expression library constructed from SW620 colosphere RNA (as detailed in the Materials and Methods). b NRIP2 expression in colorectal cancer cells. NRIP2 was detected by western blotting in colorectal cancer cells. The results showed that P1, HT29, and SW620 cells expressed NRIP2. c NRIP2 mRNA hybridization. Slides containing primary colorectal cancer tissue and normal colorectal tissue were hybridized with labeled probes for NRIP2 mRNA respectively, with a nonsense probe as a negative control. FISH analysis showed NRIP2 expression in primary colorectal cancer cells. d Expression of NRIP2 in primary colorectal cancer tissue. Colorectal cancer cells were reacted with antibodies against NRIP2 and subsequently detected by IHC staining. Normal rabbit IgG was used as a negative control. e NRIP2 increased in CD44 + CD24+ cancer cells. NRIP2 mRNA levels were determined by Taqman RT-qPCR in CD44 + CD24+ and CD44-CD24- FACS-isolated cells. NRIP2 expression increased in CD44 + CD24+ cells compared with CD44-CD24- cells; *p < 0.05 (ANOVA). f NRIP2 expression in colospheres from colorectal cancer cells. NRIP2 levels were determined by Taqman RT-qPCR in colospheres from P1, HT29, RKO, and SW620 cells under low-adhesive and serum-free conditions for 7 days. NRIP2 mRNA expression was significantly higher in colospheres than in parental cells. *p < 0.05; ***p < 0.001 (ANOVA). g NRIP2 expression in colospheres from primary colorectal cancer cells. NRIP2 levels were determined by Taqman RT-qPCR in colospheres from primary colorectal cancer samples under low-adhesive and serum-free conditions for 7 days. NRIP2 mRNA expression was significantly higher in colospheres than in their parental cells. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA). h Analysis of the relationship between NRIP2 and the colorectal cancer subtypes. The relationship between NRIP2 expression and colorectal cancer molecular typing was analyzed by the global cDNA expression GeneChip database (n = 565). NRIP2 expression was closely related to the C4-cancer stem cell (CSC) and C6-CIN colorectal cancer subtypes. C4 vs C2 or C3, all p < 0.05; C4 vs C2 or C3, all p < 0.01. i GSEA analysis. GSEA analysis from primary colorectal cancer tissues with a high level of NRIP2 expression (n = 200). Similar alterations in the mRNA expression profiles were found in cases with high levels of NRIP2 expression and in ES cells with histone methylation or iPS cells without histone methylation