Fig. 4

POU6F2-AS1 interacts with YBX1 to promote CRC cell growth and lipogenesis. (A) Schematic workflow of the biotinylated RNA pull-down assay and Mass spectrometry used for identification of POU6F2-AS1 binding proteins. (B) Venn diagram exhibiting the proteins pulled down by biotin-labeled POU6F2-AS1, and overlapping analysis with RBPDB and RBPmap database. (C) Representative YBX1 peptides binding with POU6F2-AS1 were detected by MS. (D) Western blotting of YBX1 pull downed by biotin-labeled POU6F2-AS1 was shown. (E) qRT–PCR analysis of POU6F2-AS1 enriched by YBX1 protein in SW480 cells was shown. (F) Colocalization analysis was assessed with the specific probes against POU6F2-AS1 (red) and the specific antibody against YBX1 (green). Nuclei are stained with DAPI (blue). (G) The potential interaction regions of POU6F2-AS1 with YBX1 were predicted by catRAPID website. (H) Schematic view of truncated fragments of POU6F2-AS1. (I) Western blotting analysis of YBX1 in samples pulled down by biotin-labeled full-length (F1, 2) or truncated POU6F2-AS1 (F3–F5). (J) Domain mapping of Flag-labeled full-length YBX1 (P4) or truncated YBX1 (P1–P3). (K) Western Blot analysis of Flag-tagged YBX1 constructs in SW480 cells transfected with each construct (P1: 1–129 aa, P2: 130–205 aa, P3: 206–324 aa). (L) RIP assays were performed by the antibodies against Flag, followed by POU6F2-AS1 qRT–PCR in SW480 cells. (M) CCK–8 assay in POU6F2-AS1 overexpression LoVo cells with YBX1 knockdown. (N) Colony formation assay in POU6F2-AS1 overexpression LoVo cells with YBX1 knockdown. (O) Oil Red O staining assay in POU6F2-AS1 overexpression LoVo cells with YBX1 knockdown. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001