Fig. 5

LacRNA regulates the stability of PHB2 protein. a RT-qPCR analysis of LacRNA expression and immunoblotting of PHB2 expression in 8 breast cancer cells and a normal breast cell line correspondently. The data are shown as mean ± SEM; n = 3 independent experiments. b The expression of PHB2 protein in the cytoplasmic and nuclear fractionations according to immunoblotting. c Immunoblotting of the expression of PHB2 in MDA-MB-231 and T47D cells transfected with empty vector (pWPXL-vector) or pWPXL-LacRNA. d Immunoblotting of the expression of PHB2 in MDA-MB-231 cells with LacRNA knockout by CRISPR/Cas9 technology. e Immunoblotting of the expression of PHB2 in MDA-MB-231 cells treated with the transcription inhibitor cycloheximide (CHX,100 μg/ml) for the indicated times and transfected with empty vector (pWPXL-vector) or pWPXL-LacRNA. f Immunoblotting for the protein levels of PHB2 in MDA-MB-231 cells with the proteasome inhibitor MG132 and autophagy inhibitors chloroquine (CQ) and 3-methyladenine (3MA) treated. g PHB2 autophagic degradation time in pWPXL-LacRNA and empty vector (pWPXL-vector) cells treated with the autophagy activator rapamycin for the indicated times. h Exogenous synthetic LC3-His protein was added to cell lysates of PHB2-FLAG protein-overexpressing cells (pCDH-PHB2-FLAG), and an immunoprecipitation assay with flag tag antibody was performed in the presence of sense or antisense LacRNA. Immunoblotting with a His tag antibody was used to test the immunoprecipitated LC3. i PHB2 rescue was performed after knockout of LacRNA in MDA-MB-231 cells, and transwell assays were used to assess cell migration. j Representative images of the transwell migration rescue assays in LacRNA knockout LM2 cells with additional PHB2 overexpressed. Mean ± SEM in i and j; n = 3 independent experiments; statistical significance was determined by one-way ANOVA. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Uncropped images of the blots are shown in Additional file 1: Fig. S9