Fig 2: Carfilzomib and bortezomib inhibit HER2 autophosphorylation, block ERa expression and cause cell death in ER+/HER2-amplified breast cancer cells(A–D) Carfilzomib and bortezomib induce cell death in a concentration-dependent manner. (A) and (C) Equal numbers of BT474 or MDA-MB-361 cells were seeded on 12-well culture plates and treated with the indicated carfilzomib or bortezomib concentrations. After 10 days cells were fixed and stained. (B) and (D) BT474 and MDA-MB-361 cells were cultured in the presence of the indicated carfilzomib and bortezomib concentrations for 32 hours. Western blots of protein lysates were probed with the indicated antibodies.

Fig 3: Carfilzomib and bortezomib act in concert with lapatinib to inhibit HER2 activity and to induce cell death in ER+/HER2+ breast cancer cells(A) Equal numbers of BT474 cells were seeded on 12-well culture plates and treated with the indicated lapatinib concentrations. After 10 days cells were fixed and stained (left panel). BT474 cells were cultured in the presence of the indicated lapatinib concentrations for 32 hours. Western blots of protein lysates were probed with the indicated antibodies (right panel). (B) BT474 cells were cultured in the absence or in the presence of the indicated concentrations of lapatinib, bortezomib or both drugs together (left panel) or BT474 cells were cultured in the absence or presence of the indicated concentrations of lapatinib, carfilzomib or in combination (right panel). After 30 hours cells were harvested. Western blots of the protein lysates were probed with the indicated antibodies. (C) Equal numbers of BT474 cells were seeded on 6-well culture plates and cultured either with or without the indicated lapatinib, bortezomib or carfilzomib concentrations. After 12 days cells were fixed. Macroscopic photos are shown of the fixed cell colonies for BT474 cells treated with lapatinib, bortezomib and lapatinib plus bortezomib (upper panel) and lapatinib, carfilzomib and lapatinib plus carfilzomib (lower panel). Bar 100 µm. (D) To determine the induction of cell death relative to the applied lapatinib and or bortezomib/carfilzomib concentrations equal numbers of BT474 cells were seeded on 12-well culture plates and cultured in the absence or in the presence of the indicated drug concentrations for 5 days. The percentage of SubG1 cells was evaluated using propidium iodide staining and flow cytometry. Mean values ± s.d. of three independent experiments are presented.

Fig 4: Schematic representation of anti-HER2 phage-based vaccine production.

Fig 5: IgG purified from ECTM- and ?16ECTM-immune sera impaired ERK activation in BT-474 BC cells. (A). Representative Western blot analysis of HER2 downstream signaling pathways in BT-474 cells, treated or not with trastuzumab or control-IgG or immune-IgG at the indicated concentrations for 8 h. Expression level of HER2, pHER2, AKT, pAKT, ERK, pERK was analyzed. Equal amounts of protein (20 µg) were loaded and ß-actin was used as loading control. Data are representative of a typical experiment repeated three times with similar results. (B). Densitometric quantification of HER2 and ERK expression, normalized on ß-actin, and of pERK/ERK from three independent experiments were shown; one-way ANOVA test followed by Tukey’s multiple comparison test (* p = 0.05; *** p = 0.001; **** p = 0.0001: IgG ECTM or IgG ?16ECTM vs. IgG empty; trastuzumab vs. control). (C). IgG purified from ECTM- and ?16ECTM-immune sera reduced the levels of phosphorylated RB in BT-474 cells. Upper panel: representative Western blot analysis of phosphorylated RB (Ser 780) in BT-474 cells, treated or not with 15 µg/mL trastuzumab or control-IgG or immune-IgG (30 µg/mL) for 8 h. Equal amounts of protein (20 µg) were loaded and vinculin was used as a loading control. Data are representative of a typical experiment repeated three times with similar results. Lower panel: densitometric quantification of phosphorylated RB expression, normalized on vinculin, was shown; one-way ANOVA test followed by Tukey’s multiple comparison test **** p = 0.0001: IgG ECTM or IgG ?16ECTM vs. IgG empty; trastuzumab vs. control. The original western blot data can be found in Supplementary Materials File S1 (pages 1–3).