Fig. 2.

SORLA regulates HER2 cell-surface levels and oncogenic signalling in breast cancer cells. a−c SORLA-high BT474 cells were subjected to shRNA-mediated control (shCTRL) or SORLA (shSORLA #1 and #4) silencing. SORLA-intermediate/low MDA-MB-361/JIMT-1 cells were transfected with SORLA-GFP or GFP alone. Flow cytometry analysis of cell-surface HER2 levels (a, MFI ± standard deviation (s.d.); n = 3 independent experiments), immunoblotting of total HER2 and SORLA, where α-tubulin is a loading control (b) and quantification of total HER2 protein levels relative to loading control (c) are shown (mean ± s.d.; n = 3 independent experiments; statistical analysis: unpaired Student’s t test). d Proliferation of BT474 cells after SORLA silencing with siRNAs (mean ± s.d. of n = 12, four technical replicates, three independent experiments; statistical analysis: two-way ANOVA). e Proliferation of parental, GFP control and SORLA-GFP overexpressing JIMT-1 cells (mean ± s.d. of n = 12, four technical replicates, three independent experiments; statistical analysis: two-way ANOVA). f Western blot analysis of the indicated signalling proteins (phosphorylated and total) in BT474 cells after scramble (siCTRL) and SORLA silencing (siSORLA #3 and siSORLA #4). α-tubulin is a loading control. m.#1 indicates membrane number 1 and m.#2 indicates membrane number 2 (n = 2 independent experiments). g Proliferation of MDA-MB-361 cells expressing SORLA-GFP or GFP-control after silencing with control siRNA (siCTRL) or siRNA against the 3′UTR of SORLA (siSORLA 3′UTR) (mean ± s.d. of n = 14 from four independent experiments, two-way ANOVA). h Proliferation of MDA-MB-361 cells expressing SORLA-GFP constructs (full-length, ECD-TM or TM-CD) after silencing with siCTRL or siSORLA 3′UTR (mean ± s.d. of n = 8 from two independent experiments; statistical analysis: two-way ANOVA). i Quantification of mammary DCIS tumours (Carnoy staining) 10 weeks after injection of shSORLA- and shCTRL-silenced MDA-MB-361 cells into the mammary ducts of NOD.SCID mice (box plot represents median and 25th and 75th percentiles—interquartile range; IQR—and whiskers extend to maximum and minimum values; n = 9 mice per group; statistical analysis: unpaired Student’s t test). Scale bars: 2 mm