no. on SKBR3 cells was found ( 0.001). (B) A similar synergistic cell growth inhibitory effect was determined for Lapatinib in combination with 25 M Deferiprone on BT474 cells ( 0.001). The results represent the mean values SD of three independent experiments. Image_3.tif (687K) GUID:?6B32DA3E-B39C-48CD-8D00-36EB518CEE21 Supplementary Figure 4: Uncropped Western blot images from the main text (provided as a separate PDF file). Pictures depict membranes probed with antibodies as indicated on the blots. Black boxes represent final cropped regions shown in manuscript. All top to bottom images are different exposure from the same membrane. (A) Whole, uncropped Western blots of Figure 1A. Samples order in the frames from left to right were: MDA-MB-231 cell lysate fraction (MDA cell), MDA-MB-231 cytosolic fraction (MDA cyt.), MDA-MB-231 membrane fraction (MDA mem.), HCC-1954 cell lysate (HCC cell), HCC-1954 cytosolic fraction (HCC cyt.), HCC-1954 membrane fraction (HCC mem.). (BCD) Whole, uncropped Western blots of Figure 3. Three biological replicates were loaded for each sample on the same gel. Samples order from left to right for each blot were: HCC-1954 cell lysate, MDA-MB-231 cell lysate, MCF-10A cell lysate (N=3). (E) Whole, uncropped Western blots of Figure 6A. Two biological replicates were loaded for each sample on the same gel. Samples order from left to right for each blot were: HCC-1954 cell lysate, NC-siRNA cell lysate, A-siRNA cell lysate, B-siRNA cell lysate, C-siRNA cell lysate (N=2). Presentation_1.pptx (1.6M) GUID:?30B028CC-1A03-489E-AEE1-01A03A056149 Supplementary Table 1: Complete lists of membrane proteins found in the HCC-1954 cells and MCF-10A cells (provided as a separate excel file). Combined lists of all the membrane-associated proteins found in four d-Atabrine dihydrochloride replicates of the HCC-1954 cells compared to four replicates of the MCF-10A cells along with their Normalized Areas. Bold letter proteins represent the top 30 candidate protein targets shown in the Table 1 of the main text. Table_1.xlsx (375K) GUID:?AD66C176-937E-4B96-80AA-05D2F6CFCF9F Supplementary Table 2: Complete lists of membrane proteins found in the MDA-MB-231 cells and MCF-10A cells (provided as a separate excel file). Combined lists of all the membrane-associated proteins found in four replicates of the MDA-MB-231 versus the MCF-10A cells along with their Normalized Areas. Bold letter proteins represent the top 30 candidate protein targets shown in the Table 2 of the main text. Table_2.xlsx (407K) GUID:?9B239DC8-E678-46BE-8870-A1C9E4DE8FAB Data Availability StatementThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ProteomeXchange Consortium the PRIDE partner repository with the dataset identifier PXD021819. Abstract Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC p38gamma subtypes and can serve as potential biomarkers or drug targets. We leveraged on d-Atabrine dihydrochloride the development of a membrane d-Atabrine dihydrochloride enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in.