After washes in PBST, serial dilutions of F1 or E2 were added at 4?C for 2?h and interaction revealed with an anti-human Fc antibody conjugated to HRP (1/2000; 355?ng/well)

After washes in PBST, serial dilutions of F1 or E2 were added at 4?C for 2?h and interaction revealed with an anti-human Fc antibody conjugated to HRP (1/2000; 355?ng/well). peptides and contributes to protein identification. The putative biomarkers discovered by this method require subsequent validation, for instance by immunohistochemistry. (PPTX 42 kb) 40425_2019_498_MOESM3_ESM.pptx (42K) GUID:?312F9975-62AC-4209-B415-7FD163C62D98 Additional file 4: Figure S3. Representative image of cath-D expression in TNBC biopsies. Cath-D expression was monitored by IHC using monoclonal anti-human Z-LEHD-FMK cath-D (C-5; sc-377127) antibody in TMA. Staining is prominent in breast cancer cells and is also detected in the tumor stroma. Scale bar, 100 m. (PPTX 2200 kb) 40425_2019_498_MOESM4_ESM.pptx (2.2M) GUID:?CE651B88-B444-4310-95DF-1D18B3E978DA Additional file 5: Figure S4. Generation of anti-cath-D human scFv fragments by phage display. (A) Enrichment of anti-cath-D polyclonal scFv fragments by phage display. ScFv phages specific for human mature 34+14-kDa cath-D were selected and enriched in four biopanning rounds, and analyzed by ELISA using a HRP-labeled anti-M13 antibody. BSA, negative antigen. (B) Selection of anti-cath-D monoclonal scFv fragments by ELISA. ELISA performed using bacterial culture supernatants of the best scFv clones (5 out of 400 screened clones) and recombinant human mature 34+14-kDa cath-D and 52-kDa pro-cath-D. Binding of the scFv clones to cath-D was detected with a HRP-labeled anti-Myc antibody. BSA, negative antigen; IR, irrelevant scFv from the screen. (C) Purification of the anti-human cath-D scFv fragments. His-tagged anti-cath-D scFv fragments were purified using TALON resin, resolved by 12% SDS-PAGE and stained with Coomassie blue. (D) Binding of purified anti-cath-D monoclonal scFv antibodies to human cath-D from MDA-MB-231 cells. Binding of purified anti-cath-D scFv antibodies to secreted pro-cath-D and cellular cath-D from MDA-MB-231 cells was assayed by ELISA using an anti-His HRP-conjugated antibody (left panel). BSA, negative antigen; IR, irrelevant scFv; = 3 Right panel, a whole cell lysate (10 g) and conditioned medium (80 l) from MDA-MB-231 cells were analyzed by 12% SDS-PAGE and immunoblotting using a polyclonal anti-mouse cath-D (sc-6486) antibody that cross-reacts with human cath-D (52-, 48- and 34-kDa isoforms). = 3. Right panel, whole mouse embryonic fibroblast lysate (25 g) was analyzed by 12% SDS-PAGE and immunoblotting using a polyclonal anti-mouse cath-D (sc-6486) antibody against the mouse cellular cath-D 48- and 34-kDa isoforms. = 9 per group. (PPTX 64 kb) 40425_2019_498_MOESM8_ESM.pptx (64K) GUID:?A0120614-6219-44AE-957B-B7A138EDE785 Additional file 9: Figure S8. Effect of F1 and E2 on tumor cell proliferation, apoptosis, and angiogenesis in MDA-MB-231 tumor cell xenografts. (A) Ki67 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (B) Quantification of Ki67. Percentage (mean SEM) of Ki67-positive cells relative to total cell number (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (C) Activated caspase 3 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (D) Quantification of activated caspase 3. Percentage (mean SEM) of activated caspase 3-positive pixels relative to total pixels (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (E) CD31 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (F) Quantification of CD31. Percentage (mean SEM) of CD31 cells/field (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (PPTX 1660 kb) 40425_2019_498_MOESM9_ESM.pptx (1.6M) GUID:?DC0363ED-C9A2-44DC-9857-A71C78AAFFEF Additional file 10: Figure S9. Binding of F1Fc to pro-cath-D secreted from MDA-MB-231 cells. Sandwich ELISA in which pro-cath-D from conditioned medium of MDA-MB-231 cells was added to wells pre-coated with the anti-pro-cath-D M2E8 mouse monoclonal antibody in the presence of F1Fc (1g/ml) or F1 (1g/ml). Binding of F1Fc and F1 to pro-cath-D was revealed with an anti-human Fc antibody conjugated to HRP. RTX, rituximab (negative control antibody). (PPTX 56 kb) 40425_2019_498_MOESM10_ESM.pptx (56K) GUID:?D64D249C-C68F-43F2-8495-C45127BED5EA Abstract Background Triple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC. Methods Cath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned.The anti-human cath-D antibody against 4-kDa pro-domain was kindly provided by Prof M. method used for the identification of accessible proteins in TNBC samples.The technique involves the biotinylation of TNBC biopsies by immersion in the chemically modified biotin solution. Proteins are solubilized and biotinylated protein are captured over the streptavidin materials then simply. After their enrichment, these protein can be examined using HPLC chromatographic parting, MS analysis, collection of a specific mass (peptide), and fragmentation (MS/MS). MS/MS produces a design that provides the series from the contributes and peptides to protein identification. The putative biomarkers uncovered by this technique require following validation, for example by immunohistochemistry. (PPTX 42 kb) 40425_2019_498_MOESM3_ESM.pptx (42K) GUID:?312F9975-62AC-4209-B415-7FD163C62D98 Additional document 4: Amount S3. Representative picture of cath-D appearance in TNBC biopsies. Cath-D appearance was supervised by IHC using monoclonal anti-human cath-D (C-5; sc-377127) antibody in TMA. Staining is normally prominent in breasts cancer tumor cells and can be discovered in the tumor stroma. Range club, 100 m. (PPTX 2200 kb) 40425_2019_498_MOESM4_ESM.pptx (2.2M) GUID:?CE651B88-B444-4310-95DF-1D18B3E978DA Extra file 5: Amount S4. Era of anti-cath-D individual scFv fragments by phage screen. (A) Enrichment of anti-cath-D polyclonal scFv fragments by phage screen. ScFv phages particular Z-LEHD-FMK for individual older 34+14-kDa cath-D had been chosen and enriched in four biopanning rounds, and examined by ELISA utilizing a HRP-labeled anti-M13 antibody. BSA, detrimental antigen. (B) Collection of anti-cath-D monoclonal scFv fragments by ELISA. ELISA performed using bacterial lifestyle supernatants of the greatest scFv clones (5 out of 400 screened clones) and recombinant individual mature 34+14-kDa cath-D and 52-kDa pro-cath-D. Binding from the scFv clones to cath-D was discovered using a HRP-labeled anti-Myc antibody. BSA, detrimental antigen; IR, unimportant scFv in the display screen. (C) Purification from the anti-human cath-D scFv fragments. His-tagged anti-cath-D scFv fragments had been purified using TALON resin, solved by 12% SDS-PAGE and stained with Coomassie blue. (D) Binding of purified anti-cath-D monoclonal scFv antibodies to individual cath-D from MDA-MB-231 cells. Binding of purified anti-cath-D scFv antibodies to secreted pro-cath-D and mobile cath-D from MDA-MB-231 cells was assayed by ELISA using an anti-His HRP-conjugated antibody (still left -panel). BSA, detrimental antigen; IR, unimportant scFv; = 3 Best panel, a complete cell lysate (10 g) and conditioned moderate (80 l) from MDA-MB-231 cells had been examined by 12% SDS-PAGE and immunoblotting utilizing a polyclonal anti-mouse cath-D (sc-6486) antibody that cross-reacts with individual cath-D (52-, 48- and 34-kDa isoforms). = 3. Best panel, entire mouse embryonic fibroblast lysate (25 g) was examined by 12% SDS-PAGE and immunoblotting utilizing a polyclonal anti-mouse cath-D (sc-6486) antibody against the mouse mobile cath-D 48- and 34-kDa isoforms. = 9 per group. (PPTX 64 kb) 40425_2019_498_MOESM8_ESM.pptx (64K) GUID:?A0120614-6219-44AE-957B-B7A138EDE785 Additional file 9: Figure S8. Aftereffect of F1 and E2 on tumor cell proliferation, apoptosis, and angiogenesis in MDA-MB-231 tumor cell xenografts. (A) Ki67 immunostaining. Representative pictures in tumors from CTRL- (rituximab), F1- and E2-treated mice. Range pubs, 100 m. (B) Quantification of Ki67. Percentage (mean SEM) of Ki67-positive cells in accordance with total cellular number (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (C) Activated caspase 3 immunostaining. Representative pictures in tumors from CTRL- (rituximab), F1- and E2-treated mice. Range pubs, 100 m. (D) Quantification of turned on caspase 3. Percentage (mean SEM) of turned on caspase 3-positive pixels in accordance with total pixels (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (E) Compact disc31 immunostaining. Representative pictures in tumors from CTRL- (rituximab), F1- and E2-treated mice. Range pubs, 100 m. (F) Quantification of Compact disc31. Percentage (mean SEM) of Compact disc31 cells/field (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (PPTX 1660 kb) 40425_2019_498_MOESM9_ESM.pptx (1.6M) GUID:?DC0363ED-C9A2-44DC-9857-A71C78AAFFEF Extra file 10: Amount S9. Binding of F1Fc to pro-cath-D secreted from MDA-MB-231 cells. Sandwich ELISA where pro-cath-D from conditioned moderate of MDA-MB-231 cells was put into wells pre-coated using the anti-pro-cath-D M2E8 mouse monoclonal antibody in the current presence of F1Fc (1g/ml) or F1 (1g/ml). Binding of F1Fc and F1 to pro-cath-D was uncovered with an anti-human Fc antibody conjugated to HRP. RTX, rituximab (detrimental control antibody). (PPTX 56 kb) 40425_2019_498_MOESM10_ESM.pptx (56K) GUID:?D64D249C-C68F-43F2-8495-C45127BED5EA Abstract History.Cath-D expression was monitored by IHC using monoclonal anti-human cath-D (C-5; sc-377127) antibody in TMA. their enrichment, these proteins could be examined using HPLC chromatographic separation, MS analysis, collection of a specific mass (peptide), and fragmentation (MS/MS). MS/MS produces a design that delivers the series from the peptides and plays a part in protein id. The putative biomarkers uncovered by this technique require following validation, for example by immunohistochemistry. (PPTX 42 kb) 40425_2019_498_MOESM3_ESM.pptx (42K) GUID:?312F9975-62AC-4209-B415-7FD163C62D98 Additional document 4: Amount S3. Representative picture of cath-D appearance in TNBC biopsies. Cath-D appearance was supervised by IHC using monoclonal anti-human cath-D (C-5; sc-377127) antibody in TMA. Staining is normally prominent in breasts cancer tumor cells and can be discovered in the tumor stroma. Range club, 100 m. (PPTX 2200 kb) 40425_2019_498_MOESM4_ESM.pptx (2.2M) GUID:?CE651B88-B444-4310-95DF-1D18B3E978DA Extra file 5: Amount S4. Era of anti-cath-D individual scFv fragments by phage screen. (A) Enrichment of anti-cath-D polyclonal scFv fragments Z-LEHD-FMK by phage screen. ScFv phages particular for individual older 34+14-kDa cath-D had been chosen and enriched in four biopanning rounds, and examined by ELISA utilizing a HRP-labeled anti-M13 antibody. BSA, detrimental antigen. (B) Collection of anti-cath-D monoclonal scFv fragments by ELISA. ELISA performed using bacterial lifestyle supernatants of the greatest scFv clones (5 out of 400 screened clones) and recombinant individual mature 34+14-kDa cath-D and 52-kDa pro-cath-D. Binding from the scFv clones to cath-D was discovered using a HRP-labeled anti-Myc antibody. BSA, detrimental antigen; IR, unimportant scFv from the screen. (C) Purification of the anti-human cath-D scFv fragments. His-tagged anti-cath-D scFv fragments were purified using TALON resin, resolved by 12% SDS-PAGE and stained with Coomassie blue. (D) Binding of purified anti-cath-D monoclonal scFv antibodies to human cath-D from MDA-MB-231 cells. Binding of purified anti-cath-D scFv antibodies to secreted pro-cath-D and cellular cath-D from MDA-MB-231 cells was assayed by ELISA using an anti-His HRP-conjugated antibody (left panel). BSA, unfavorable antigen; IR, irrelevant scFv; = 3 Right panel, a whole cell lysate (10 g) and conditioned medium (80 l) from MDA-MB-231 cells were analyzed by 12% SDS-PAGE and immunoblotting using a polyclonal anti-mouse cath-D (sc-6486) antibody that cross-reacts with human cath-D (52-, 48- and 34-kDa isoforms). = 3. Right panel, whole mouse embryonic fibroblast lysate (25 g) was analyzed by 12% SDS-PAGE and immunoblotting using a polyclonal anti-mouse cath-D (sc-6486) antibody against the mouse cellular cath-D 48- and 34-kDa isoforms. = 9 per group. (PPTX 64 kb) 40425_2019_498_MOESM8_ESM.pptx (64K) GUID:?A0120614-6219-44AE-957B-B7A138EDE785 Additional file 9: Figure S8. Effect of F1 and E2 on tumor cell proliferation, apoptosis, and angiogenesis in MDA-MB-231 tumor cell xenografts. (A) Ki67 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (B) Quantification of Ki67. Percentage (mean SEM) of Ki67-positive cells relative to total cell number (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (C) Activated caspase 3 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (D) Quantification of activated caspase 3. Percentage (mean SEM) of activated caspase 3-positive pixels relative to total pixels (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (E) CD31 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (F) Quantification of CD31. Percentage (mean SEM) of CD31 cells/field (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (PPTX 1660 kb) 40425_2019_498_MOESM9_ESM.pptx (1.6M) GUID:?DC0363ED-C9A2-44DC-9857-A71C78AAFFEF Additional file 10: Physique S9. Binding of F1Fc to pro-cath-D secreted from MDA-MB-231 cells. Sandwich ELISA in which pro-cath-D from conditioned medium of MDA-MB-231 cells was added to wells pre-coated with the anti-pro-cath-D M2E8 mouse monoclonal antibody in the presence of F1Fc (1g/ml) or F1 (1g/ml). Binding of F1Fc and F1 to pro-cath-D was revealed with an.Right panel, whole mouse embryonic fibroblast lysate (25 g) was analyzed by 12% SDS-PAGE and immunoblotting using a polyclonal anti-mouse cath-D (sc-6486) antibody against the mouse cellular cath-D 48- and 34-kDa isoforms. a particular mass (peptide), and fragmentation (MS/MS). MS/MS yields a pattern that delivers the sequence of the peptides and contributes to protein identification. The putative biomarkers discovered by this method require subsequent validation, for instance by immunohistochemistry. (PPTX 42 kb) 40425_2019_498_MOESM3_ESM.pptx (42K) GUID:?312F9975-62AC-4209-B415-7FD163C62D98 Additional file 4: Physique S3. Representative image of cath-D expression in TNBC biopsies. Cath-D expression was monitored by IHC using monoclonal anti-human cath-D (C-5; sc-377127) antibody in TMA. Staining is usually prominent in breast malignancy cells and is also detected in the tumor stroma. Scale bar, 100 m. (PPTX 2200 kb) 40425_2019_498_MOESM4_ESM.pptx (2.2M) GUID:?CE651B88-B444-4310-95DF-1D18B3E978DA Additional file 5: Physique S4. Generation of anti-cath-D human scFv fragments by phage display. (A) Enrichment of anti-cath-D polyclonal scFv fragments by phage display. ScFv phages specific for human mature 34+14-kDa cath-D were selected and enriched in four biopanning rounds, and analyzed by ELISA using a HRP-labeled anti-M13 antibody. BSA, unfavorable antigen. (B) Selection of anti-cath-D monoclonal scFv fragments by ELISA. ELISA performed using bacterial culture supernatants of the best scFv clones (5 out of 400 screened clones) and recombinant human mature 34+14-kDa cath-D and 52-kDa pro-cath-D. Binding of the scFv clones to cath-D was detected with a HRP-labeled Rabbit polyclonal to ZMAT5 anti-Myc antibody. BSA, unfavorable antigen; IR, irrelevant scFv from the screen. (C) Purification of the anti-human cath-D scFv fragments. His-tagged anti-cath-D scFv fragments were purified using TALON resin, resolved by 12% SDS-PAGE and stained with Coomassie blue. (D) Binding of purified anti-cath-D monoclonal scFv antibodies to human cath-D from MDA-MB-231 cells. Binding of purified anti-cath-D scFv antibodies to secreted pro-cath-D and cellular cath-D from MDA-MB-231 cells was assayed by ELISA using an anti-His HRP-conjugated antibody (left panel). BSA, unfavorable antigen; IR, irrelevant scFv; = 3 Right panel, a whole cell lysate (10 g) and conditioned medium (80 l) from MDA-MB-231 cells were analyzed by 12% SDS-PAGE and immunoblotting using a polyclonal anti-mouse cath-D (sc-6486) antibody that cross-reacts with human cath-D (52-, 48- and 34-kDa isoforms). = 3. Right panel, whole mouse embryonic fibroblast lysate (25 g) was analyzed by 12% SDS-PAGE and immunoblotting using a polyclonal anti-mouse cath-D (sc-6486) antibody against the mouse cellular cath-D 48- and 34-kDa isoforms. = 9 per group. (PPTX 64 kb) 40425_2019_498_MOESM8_ESM.pptx (64K) GUID:?A0120614-6219-44AE-957B-B7A138EDE785 Additional file 9: Figure S8. Effect of F1 and E2 on tumor cell proliferation, apoptosis, and angiogenesis in MDA-MB-231 tumor cell xenografts. (A) Ki67 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (B) Quantification of Ki67. Percentage (mean SEM) of Ki67-positive cells relative to total cell number (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (C) Activated caspase 3 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (D) Quantification of activated caspase 3. Percentage (mean SEM) of activated caspase 3-positive pixels relative to total pixels (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (E) CD31 immunostaining. Representative images in tumors from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 m. (F) Quantification of CD31. Percentage (mean SEM) of CD31 cells/field (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (PPTX 1660 kb) 40425_2019_498_MOESM9_ESM.pptx (1.6M) GUID:?DC0363ED-C9A2-44DC-9857-A71C78AAFFEF Additional file 10: Physique S9. Binding of F1Fc to pro-cath-D secreted from MDA-MB-231 cells. Sandwich ELISA in which pro-cath-D from conditioned medium of MDA-MB-231 cells was added to wells pre-coated with the anti-pro-cath-D M2E8 mouse monoclonal antibody in the presence of F1Fc (1g/ml) or F1 (1g/ml). Binding of F1Fc and F1 to pro-cath-D was revealed with an anti-human Fc antibody conjugated to HRP. RTX, rituximab (unfavorable control antibody). (PPTX 56 kb) 40425_2019_498_MOESM10_ESM.pptx (56K) GUID:?D64D249C-C68F-43F2-8495-C45127BED5EA Abstract Background Triple-negative breast malignancy (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment.D3417227). for instance by immunohistochemistry. (PPTX 42 kb) 40425_2019_498_MOESM3_ESM.pptx (42K) GUID:?312F9975-62AC-4209-B415-7FD163C62D98 Additional file 4: Physique S3. Representative image of cath-D expression in TNBC biopsies. Cath-D expression was monitored by IHC using monoclonal anti-human cath-D (C-5; sc-377127) antibody in TMA. Staining is usually prominent in breast malignancy cells and is also detected in the tumor stroma. Scale bar, 100 m. (PPTX 2200 kb) 40425_2019_498_MOESM4_ESM.pptx (2.2M) GUID:?CE651B88-B444-4310-95DF-1D18B3E978DA Additional file 5: Physique S4. Generation of anti-cath-D human scFv fragments by phage display. (A) Enrichment of anti-cath-D polyclonal scFv fragments by phage display. ScFv phages particular for human being adult 34+14-kDa cath-D had been chosen and enriched in four biopanning rounds, and examined by ELISA utilizing a HRP-labeled anti-M13 antibody. BSA, adverse antigen. (B) Collection of anti-cath-D monoclonal scFv fragments by ELISA. ELISA performed using bacterial tradition supernatants of the greatest scFv clones (5 out of 400 screened clones) and recombinant human being mature 34+14-kDa cath-D and 52-kDa pro-cath-D. Binding from the scFv clones to cath-D was recognized having a HRP-labeled anti-Myc antibody. BSA, adverse antigen; IR, unimportant scFv through the display. (C) Purification from the anti-human cath-D scFv fragments. His-tagged anti-cath-D scFv fragments had been purified using TALON resin, solved by 12% SDS-PAGE and stained with Coomassie blue. (D) Binding of purified anti-cath-D monoclonal scFv antibodies to human being cath-D from MDA-MB-231 cells. Binding of purified anti-cath-D scFv antibodies to secreted pro-cath-D and mobile cath-D from MDA-MB-231 cells was assayed by ELISA using an anti-His HRP-conjugated antibody (remaining -panel). BSA, adverse antigen; IR, unimportant scFv; = 3 Best panel, a complete cell lysate (10 g) and conditioned moderate (80 l) from MDA-MB-231 cells had been examined by 12% SDS-PAGE and immunoblotting utilizing a polyclonal anti-mouse cath-D (sc-6486) antibody that cross-reacts with human being cath-D (52-, 48- and 34-kDa isoforms). = 3. Best panel, entire mouse embryonic fibroblast lysate (25 g) was examined by 12% SDS-PAGE and immunoblotting utilizing a polyclonal anti-mouse cath-D (sc-6486) antibody against the mouse mobile cath-D 48- and 34-kDa isoforms. = 9 per group. (PPTX 64 kb) 40425_2019_498_MOESM8_ESM.pptx (64K) GUID:?A0120614-6219-44AE-957B-B7A138EDE785 Additional file 9: Figure S8. Aftereffect of F1 and E2 on tumor cell proliferation, apoptosis, and angiogenesis in MDA-MB-231 tumor cell xenografts. (A) Ki67 immunostaining. Representative pictures in Z-LEHD-FMK tumors from CTRL- (rituximab), F1- and E2-treated mice. Size pubs, 100 m. (B) Quantification of Ki67. Percentage (mean SEM) of Ki67-positive cells in accordance with total cellular number (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (C) Activated caspase 3 immunostaining. Representative pictures in tumors from CTRL- (rituximab), F1- and E2-treated mice. Size pubs, 100 m. (D) Quantification of triggered caspase 3. Percentage (mean SEM) of turned on caspase 3-positive pixels in accordance with total pixels (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (E) Compact disc31 immunostaining. Representative pictures in tumors from CTRL- (rituximab), F1- and E2-treated mice. Size pubs, 100 Z-LEHD-FMK m. (F) Quantification of Compact disc31. Percentage (mean SEM) of Compact disc31 cells/field (= 9 for rituximab (CTRL); = 9 for F1; = 9 for E2). (PPTX 1660 kb) 40425_2019_498_MOESM9_ESM.pptx (1.6M) GUID:?DC0363ED-C9A2-44DC-9857-A71C78AAFFEF Extra file 10: Shape S9. Binding of F1Fc to pro-cath-D secreted from MDA-MB-231 cells. Sandwich ELISA where pro-cath-D from conditioned moderate of MDA-MB-231 cells was put into wells pre-coated using the anti-pro-cath-D M2E8 mouse monoclonal antibody in the current presence of F1Fc (1g/ml) or F1 (1g/ml). Binding of F1Fc and F1 to pro-cath-D.