Fig. 7

RP11-296E3.2 acts as an important molecular chaperone for YBX1 that functions to promote CRC cell proliferation and metastasis by activating STAT3 transcription. A Colony formation and B Transwell assays (scale bars: 100 μm) using HCT116 cells showed that RP11-296E3.2 downregulation weakened the effects of YBX1 on cell proliferation, metastasis and invasion. C Histogram showing the counts of proliferating and migrated cells. D Colony formation and E Transwell assays (scale bars: 100 μm) using RKO cells showed that RP11-296E3.2 downregulation weakened the effects of YBX1 on cell proliferation, metastasis and invasion. F Histogram showing the counts of proliferating and migrated cells. G WB analysis of YBX1 expression in HCT116 cells transduced with the sh-RP11-296E3.2 vector and control vector. H qRT‒PCR analysis of RP11-296E3.2 expression in HCT116 cells transduced with the YBX1 overexpression vector and control vector. I The altered mRNA levels of STAT3 were confirmed by qRT-PCR in HCT116 and RKO cells transduced with the indicated shRNAs. J The STAT3 binding site promoter was subjected to a luciferase reporter assay in HCT116 cells with YBX1 overexpression with or without knockdown of RP11-296E3.2. K ChIP assays for the enrichment of YBX1 at the STAT3 promoter in HCT116 cells with YBX1 overexpression with or without knockdown of RP11-296E3.2. L The RNA pulldown assay showed that RP11-296E3.2 could not interact with STAT3. M The IP assay showed that YBX1 could interact with STAT3 and that silencing of RP11-296E3.2 did not impact the capacity of YBX1 to bind to STAT3. N The altered levels of STAT3 and p-STAT3 were confirmed by WB analysis in HCT116 and RKO cells transduced with the indicated shRNAs. The data are presented as the means ± SDs. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test for multiple comparisons. All experiments were performed with at least three biological duplicates (n = 3) for each group. *P < 0.05, **P < 0.01, ***P < 0.001