Fig. 6

The LacRNA-PHB2 complex is a negative regulator of Myc signaling. a GSEA results of the Myc targets set with RNA-sequencing data after LacRNA activation. b RT-qPCR analyses of c-Myc and c-Myc target genes in BT549 cells with LacRNA activation by the CRISPR/dead-Cas9 technology. Mean ± SEM; n = 3 independent experiments. P values were determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. c Immunoblotting of the expression of c-Myc and c-Myc downstream genes (CDC45 and MAD2L1) in MDA-MB-231 and T47D cells with LacRNA activation by the CRISPR/dead-Cas9 technology. d Rescue CHX chase assays were conducted using MDA-MB-231 cells transfected with pWPXL-vector, pWPXL-LacRNA, and siPHB2 to measure c-Myc stability. Representative images of immunoblotting analyses examining c-Myc levels in MDA-MB-231 cells at various treatment time points are shown. e Cells were transfected with pcDNA3.1-PHB2 and His-tagged Ub plasmids for 48 h. The cell lysates were immunoprecipitated (IP) with either control IgG or His antibody and immunoblotted with c-Myc-specific antibody. c-Myc and GAPDH served as the loading controls. f Immunoprecipitation to detect the association between PHB2 and c-Myc. The pcDNA3.1-Flag-PHB2 plasmid and pcDNA3.1-HA-c-Myc plasmid were cotransfected into MDA-MB-231 cells. Immunoblotting of the Flag and HA tags. g Immunoblotting for c-Myc with biotinylated sense LacRNA and anti-sense-LacRNA using streptavidin RNA pulldown assays. h Representative data from immunohistochemistry staining of c-Myc and PHB2 in orthotopic transplanted tumors obtained from NOD/SCID mice after injection with vector pWPXL-LacRNA and control pWPXL-vector LM2 cells. Uncropped images of the blots are shown in Additional file 1: Fig. S9