Fig 2: Induction of p-AKT expression on paclitaxel treatment is attenuated by HER-2 knockdown. Endometrial cancer Ishikawa (A) and HEC1A cells (B) or immortalised EM-E6/E7/TERT cells (C) were incubated with siRNA against HER-2 (siHER-2) or scrambled siRNA (siNC) for 48 h. Cells were then treated with 10 nM of paclitaxel for an additional 48 h. Cell lysates were prepared and western blot analyses were performed with antibodies against p-AKT. NC: negative control sample treated with scrambled siRNA. A representative image of the bands is shown. Bband intensity was measured using NIH image and is represented as the relative amount of p-AKT after normalisation against ß-actin expression. Each experiment was performed in triplicate in three independent experiments. Columns, mean; bars, ±s.d. *P<0.05.

Fig 3: Schematic diagram showing targeted inhibition of the ERBB-c-MET axis to prevent BC metastasis in an orthotopic ERBB2+ mouse model. (A) In vitro enrichment of the SKBrM3 cell line using a Boyden chamber. (B) Orthotopic implantation of SKBrM3+ cells in fourth mammary fat pads of female nude mice. (C) Incidences of metastasis in distant organs including brain, bone, liver, and lung. (D) Combination treatment of NER and CBZ after 3 weeks of implantation. (E,F) Effect of combination therapy on primary tumor growth and metastasis.

Fig 4: Chemoprevention of HER2-positive breast cancer by targeting theTXA2 pathway. a TBXAS1 is up-regulated in HER2-driven mammary tumorigenesis in an MMTV-Her2/neu transgenic mouse mammary tumor model. Immunohistochemical staining of TBXAS1 in mouse mammary tumors or normal mammary tissues. Original magnification: 200×. b Switching off TXA2 biosynthesis attenuates HER2-driven breast tumorigenesis. MMTV-HER2/neu transgenic mice were treated with EPA for a total of 60 weeks. c Effects of EPA intake on circulating TXA2 and PGE2 levels. d Effects of EPA intake on TBXAS1 or TBXA2R in the MMTV-neu mammary glands and tumors. Because the FVB background of MMTV-Her2/neu transgenic mice, matched non-transgenic FVB mice were used as a baseline to minimize the genetic differences. Production of serum PGs was measured by ELISA as described in “Materials and Methods”. Data are presented as mean values ± S.E.M. The asterisks indicate a significant difference among groups (*p < 0.05, ***p < 0.001)

Fig 5: Kinase Specificity of HECTD3(A) Western blot of endogenous CRAF in HEK293 cell lysates following 24-hr treatment with AUY922. Substantial degradation is observed in cells transfected with control siRNA, but CRAF levels are stabilized in cells transfected with siRNA against HECTD3. Both immunoreactive bands to the HECTD3 antibody are knocked down by the siRNA; see below. GAPDH levels provide a loading control.(B) Western blots of known HSP90 protein kinase clients in HEK293 lysates and their response to treatment of cells with AUY922. Levels of ErbB2, MASTL, CRAF, LKB1, and CDK4, but not BRAF or PDK1, decrease in response to HSP90 inhibition.(C) MASTL, LKB1, and CRAF degradation following AUY922 treatment is reduced in HEK293 cells transfected with siRNA to HECTD3 compared to a control siRNA. Knockdown of HECTD3 had no clear effect on AUY922-triggered degradation of ErbB2 or CDK4. The panel is a montage of three separate experiments; GAPDH is a loading control for each.(D) Western blot of eYFP-CRAF immunoprecipitated from cell lysates of HEK293 cells treated with increasing doses of AUY922 post-lysis. Ubiquitylation of eYFP-CRAF can be enhanced in HEK293 cell lysates, as well as in intact cells, by the addition of AUY922 in a dose-dependent fashion.