Fig 2: Levels of HER expression in breast cancer cell lines with acquired resistance to anti-oestrogen therapy. Representative Western blot data obtained from whole-cell lysates (A) separated by SDS-PAGE and immunoblotted onto PVDF membranes before immunodetection (n = 4). The head and neck cancer HN5 cell line HER1 + ve and breast cancer SKBR3 cell line HER2 + ve were used as positive controls. (B) Expression levels of parental cells (set at 1.0) were compared with the resistant variant using the following paired t-test levels of statistical significance for densitometric scans: * p < 0.05; ** p < 0.001; *** p < 0.0001.

Fig 3: The effect of rhPEPD and rhPEPDG278D on ERBB1 and ERBB1-ERBB2 heterodimer interaction. [A] CHO-K1/ERBB2 cells were transfected with human ERBB1 for 24 h and then treated with rhPEPD (5 nM), followed by immunoblotting. [B] 32D/ERBB1 cells were treated with rhPEPD or rhPEPDG278D, followed by immunoblotting. [C] 32D/ERBB1 cells were treated with vehicle, rhPEPD or rhPEPDG278D (5 nM), and then treated with cross-linking agent BS3 (2 mM, 30 min); cell lysates were immunoblotted for ERBB1. [D] CHO-K1/ERBB2 cells were treated with solvent, rhPEPD or rhPEPDG278D (each at 5 nM) for 1 h. Cell lysates were pulled down with an ERBB2 antibody, followed by immunoblotting. Sample loading was adjusted to contain an equal amount of ERRB2. An example of immunoblot without adjustment of ERBB2 content is shown in Supplementary Fig. 7. [E] 32D/ERBB1 cells were transfected with human SRC for 48 h and then treated with rhPEPD or rhPEPDG278D (5 nM), followed by immunoblotting. [F] 32D/ERBB1 cells were transfected with human SRC for 48 h and then treated with solvent, rhPEPD or rhPEPDG278D (each at 5 nM) for 1 h. Cell lysates were immunoprecipitated using an ERBB1 antibody, followed by immunoblotting. [G & H] CHO-K1/ERBB2 cells were transfected with human ERBB1 for 24 h and then treated with solvent, rhPEPD or rhPEPDG278D (each at 5 nM) for 1 h, with or without pretreatment with EGF (20 nM, 15 min). Cell lysates were immunoprecipitated using an ERBB2 antibody or an ERBB1 antibody, followed by immunoblotting. For the immunoblots shown in G, sample loading was adjusted to contain an equal amount of ERBB2.

Fig 4: VPA significantly down-regulates the expression of ErbB family members EGFR, ErbB2, and ErbB3 and modulates the activation of downstream Akt and MAPK signalings. HPAF-II and MPanc96 cells untreated or treated with VPA (0.5 mmol/L) for 24 h or 48 h were harvested and subjected to western blot analyses with specific antibodies directed against EGFR, ErbB2, ErbB3, P-Akt, Akt, P-MAPK, MAPK, P-STAT3, STAT3, RAS or ß-actin. The densitometry analyses of EGFR, ErbB2, and ErbB3 signals were shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0

Fig 5: HER1 and HER2 expression with and without tyrosine kinase inhibitor (TKI) treatment in parental MCF-7 and MCF-7-TR (tamoxifen-resistant) and T47D and T47D-FR (fulvestrant-resistant) breast cancer cell lines using confocal fluorescence microscopy. Cells were treated with 5 µM lapatinib and incubated for 48 h. TO-PRO-3 (blue) was used to label nuclei, and HER1 (A) and HER2 (B) antibody reactions were conjugated to Alexa-Fluor 488 secondary antibody. Images are representative of at least three separate experiments. Settings for the microscope were maintained as consistently as possible from one experiment to another.