Journal: Journal of Experimental & Clinical Cancer Research : CR
Article Title: The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer
doi: 10.1186/s13046-024-03099-4
Figure Lengend Snippet: Analyzing bioactivity of monoclonal anti-PRLR antibodies. ( A ) Binding of mAbs on recombinant PRLR was determined by ELISA. The ELISA plate was pre-coated with recombinant PRLR protein. Serial diluted mAbs were added to the wells as primary antibodies. ( B ) Binding of mAbs on T47D was determined by flowcytometry. Indicated mAbs were added to the cells as primary antibodies. After that, antibodies bound on cell membranes were detected by anti-human Fc-APC antibody. ( C ) Flowcytometry was used to determine internalization of PRLR-targeting mAbs. The cells were cultured with indicated antibody on ice for 60 min to saturate the cell membranes with antibody. Subsequently, cells were transferred to 37℃ to start the internalization. Antibodies left on cell membranes under 37℃ were detected by anti-human-Fc-APC at 0 h and 1 h. Internalization (%) was calculated by [MFI (0 h) – MFI (1 h)]/MFI (0 h). ( D ) Western blot detecting p-ERK (T202/T204), ERK, p-ERα (Ser118), ERα, p-STAT3 (Y705), STAT3, p-STAT5 (Y694), STAT5 and β-actin (loading control) in T47D cells stimulated by PRL in the presence of indicated PRLR-targeting mAbs. Cells were starved in DMEM devoid of FBS for 24 h prior to activation of PRL for 15 min. ( E ) Cell viability of T47D cells was determined by CCK8 in the presence of PRL with indicated PRLR-targeting mAbs. Cells were cultured for 72 h before viability was tested. Viability of cells without any treatment was set as 100%. ( F ) Viability of T47D spheroid was determined by Celltiter-glo in the presence of PRL or N8 mAb. Left: the image of spheroids in indicated groups. Right: Curve of the viability of T47D spheroids to tamoxifen concentration. Viability of T47D spheroid treated without any treatment (PBS/IgG/0µM tamoxifen) was set as 100%. ( G ) Cell viability of MCF7-TAMR cells was determined when overexpression of PRL was induced (Left) or N8 mAb was present (Right). Cell viability of MCF7-TAMR cells without any treatment (0µM tamoxifen/0µg/ml N8) was set as 100%
Article Snippet: Then, the tumor sections or microarrays were blocked with 3% bovine serum albumin in PBS for 30 min and incubated with anti-human PRLR rabbit antibody (ab170935, Abcam, England) or anti-ki67 rabbit antibody (ab15580, Abcam, England) at 4 °C for overnight.
Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Activation Assay, Concentration Assay, Over Expression
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![Analyzing bioactivity of monoclonal <t>anti-PRLR</t> antibodies. ( A ) Binding of mAbs on recombinant PRLR was determined by ELISA. The ELISA plate was pre-coated with recombinant PRLR protein. Serial diluted mAbs were added to the wells as primary antibodies. ( B ) Binding of mAbs on T47D was determined by flowcytometry. Indicated mAbs were added to the cells as primary antibodies. After that, antibodies bound on cell membranes were detected by anti-human Fc-APC antibody. ( C ) Flowcytometry was used to determine internalization of PRLR-targeting mAbs. The cells were cultured with indicated antibody on ice for 60 min to saturate the cell membranes with antibody. Subsequently, cells were transferred to 37℃ to start the internalization. Antibodies left on cell membranes under 37℃ were detected by anti-human-Fc-APC at 0 h and 1 h. Internalization (%) was calculated by [MFI (0 h) – MFI (1 h)]/MFI (0 h). ( D ) Western blot detecting p-ERK (T202/T204), ERK, p-ERα (Ser118), ERα, p-STAT3 (Y705), STAT3, p-STAT5 (Y694), STAT5 and β-actin (loading control) in T47D cells stimulated by PRL in the presence of indicated PRLR-targeting mAbs. Cells were starved in DMEM devoid of FBS for 24 h prior to activation of PRL for 15 min. ( E ) Cell viability of T47D cells was determined by CCK8 in the presence of PRL with indicated PRLR-targeting mAbs. Cells were cultured for 72 h before viability was tested. Viability of cells without any treatment was set as 100%. ( F ) Viability of T47D spheroid was determined by Celltiter-glo in the presence of PRL or N8 mAb. Left: the image of spheroids in indicated groups. Right: Curve of the viability of T47D spheroids to tamoxifen concentration. Viability of T47D spheroid treated without any <t>treatment</t> <t>(PBS/IgG/0µM</t> tamoxifen) was set as 100%. ( G ) Cell viability of MCF7-TAMR cells was determined when overexpression of PRL was induced (Left) or N8 mAb was present (Right). Cell viability of MCF7-TAMR cells without any treatment (0µM tamoxifen/0µg/ml N8) was set as 100%](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8579/pmc11188579/pmc11188579__13046_2024_3099_Fig4_HTML.jpg)
