ErbB3 gene expression correlates with BRCA patient survival and chemotherapy response. (A) ErbB3 mRNA expression levels as log2-transformed transcripts per million (TPM) in tumor or corresponding normal tissue from TCGA BRCA tumor vs TCGA normal + GTEx normal datasets were retrieved from the GEPIA2 portal. The data in the bottom indicate the number (N) of tissue samples analyzed in each group, and the differential expression analysis was performed with one-way ANOVA, using disease state as variable for calculating differential expression. Red, *P ≤ 0.01. (B) BRCA patient overall survival was calculated after sample stratification and compared with the Kaplan–Meier plotter database by auto select cutoff, based on the low and high expression level of ErbB3 mRNA. (C) BRCA patients were classified as chemotherapy nonresponders or responders based on the relapse-free survival at 5 years after treatment. The diagram presents median ErbB3 mRNA expression values, along with SEM and minimal and maximal values, as explained in the table below the graph, which also presents the number (N) of analyzed patients. Comparisons were performed using the Mann–Whitney U test (*P ≤ 0.05). (D) Representative protein expression of ErbB3 and GAPDH (as a loading control) in the cell extract of MCF7 transiently transfected with control siRNA (siCtrl) or siRNA targeting ErbB3 (#2). (E) Confocal immunofluorescence imaging of traced internalized Integrin β1 in MCF7 cells transiently transfected with control siRNA (siCtrl) or siErbB3#2. Integrin β1 that was traced for the indicated times after a 15-min internalization step. (F) RT-qPCR analysis of ErbB3 mRNA levels in MCF7 cells transiently transfected with siRNAs targeting ErbB3 (#2). Values represent fold change of ErbB3 mRNA expression relative to GAPDH. Data are presented as mean values of three biological replicates ± SEM, each in technical duplicates, and P values are shown based on unpaired Student’s t test. P values: ****P ≤ 0.0001. Scale bar, 50 µM (E).