Journal: European Journal of Histochemistry : EJH

Article Title: Seasonal patterns of prolactin, prolactin receptor, and STAT5 expression in the ovaries of wild ground squirrels ( Citellus dauricus Brandt)

doi: 10.4081/ejh.2023.3825

Figure Lengend Snippet: Types of immunohistochemistry primary antibodies.

Article Snippet: PRLR , 1:200 , bs-6445R , Bioss Biotechnology , Beijing, China.

Techniques: Immunohistochemistry

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 1:200 anti-PRLR-APC antibody (10,278-R204-A, Sino Biological, China) was added to the cells.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Activation Assay, Concentration Assay, Over Expression

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: N8-PE24 immunotoxin demonstrated rapid internalization into cells and efficiently induced cell apoptosis. ( A ) Diagram of the construction of N8-PE24 immunotoxin. Int-N: N-terminal fragment of intein. Int-C: C-terminal fragment of intein. ( B ) SDS-PAGE analysis of N8-PE24. NR: non-reduced sample. R: reduced sample. ( C ) The internalization rate of N8-PE24 immunotoxin was determined by flowcytometry. After keeping the cells under 37℃ for indicated time, N8-PE24 left on cell membrane was detected by Anti-Fab-APC secondary antibody. ( D ) The internalization of pHrodo-red-labelled N8-PE24 was analyzed under fluorescence microscope. The fluorescence was visualized after culturing the cells with pHrodo-red-labelled N8-PE24 for 4 h. ( E ) PRLR level on T47D, MCF7, MCF7-TAMR cells was analyzed by flowcytometry. The PRLR was detected by anti-PRLR-APC antibody. ( F ) Apoptosis of T47D and MCF7-TAMR cells induced by N8-PE24 was analyzed by Annexin V-FITC/PI-PE staining and flowcytometry. ( G ) Dosage and treatment schedule for MCF7-TAMR xenograft model. Female SPF grade NOD/SCID mice aged 6 weeks were implanted s.c with ten million cells on day 0. When the tumor reached a volume of 100 mm 3 , treatment began. ( H ) Tumor growth curve of MCF7-TAMR xenografts treated with or without N8-PE24 on NOD/SCID mice (12 mice in each group). ( I ) HE analysis of organs from mice treated with indicated drugs

Article Snippet: Then 1:200 anti-PRLR-APC antibody (10,278-R204-A, Sino Biological, China) was added to the cells.

Techniques: SDS Page, Membrane, Fluorescence, Microscopy, Staining

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: N8-PE24 combined with tamoxifen or paclitaxel could inhibit 231-PRLR breast cancer xenograft. ( A ) PRLR IHC analysis of tumor and adjacent normal tissues from a TNBC patient. ( B ) Flowcytometry analysis of PRLR on MDA-MB-231 cells treated with or without tamoxifen. 2.5µM tamoxifen was added to cells for 2 days before analysis. Anti-PRLR-APC antibody was used for staining. ( C ) Flowcytometry analysis of PRLR on 231-PRLR cells. Isotype-APC (red) and anti-PRLR-APC antibody (blue) were used for staining. ( D ) Western blot determining PRLR protein level in 231-PRLR cells when tamoxifen was present or not. 2.5µM tamoxifen was added to cells for 2 days before cell were collected and lysed. Cell lysates were then probed by anti-PRLR antibody, ( E ) Evaluation of cell viability by CCK8 assay to determine inhibition effect of N8-PE24 when tamoxifen was present or not on 231-PRLR BC. Viability of 231-PRLR cells treated without any reagents (0µM tamoxifen and 0 µg/ml N8-PE24) was set as 100%. ( F ) Dosage and treatment schedule for 231-PRLR xenograft model. Female SPF grade Balb/c nude mice aged 6 weeks were implanted s.c with five million cells on day 0. When the tumor reached a volume of 100 mm 3 , treatment began. ( G ) Tumor growth curve of 231-PRLR xenografts treated with indicated drugs on nude mice. Tumor volume was calculated as (long diameter (mm) × short diameter (mm) × short diameter (mm))/2. ( H ) Tumor image of 231-PRLR xenografts treated with indicated drugs. ( I ) IHC analysis of Ki67 and PRLR of 231-PRLR xenografts in different groups

Article Snippet: Then 1:200 anti-PRLR-APC antibody (10,278-R204-A, Sino Biological, China) was added to the cells.

Techniques: Staining, Western Blot, CCK-8 Assay, Inhibition

Journal:

Article Title: Biological Significance of Prolactin in Gynecological Cancers

doi: 10.1158/0008-5472.CAN-08-4652

Figure Lengend Snippet: A. RT-PCR analysis. Total RNA was isolated from normal pituitary tissue (Pit), normal endometrium (Normal), non-tumor adjacent tissue surrounding endometrial (Endo) and ovarian tumors (NAT), and endometrial and ovarian tumors (Tumor). P-proximal transcript, expected size of 617bp (red rectangle); D-distal transcript, expected size of 780bp (blue rectangle). B. Prolactin expression by endometrial and ovarian carcinoma. Ovarian carcinoma cells, OVCAR3 and SKOV3, and endometrial carcinoma cells, HEC-1A, AN3 CA, and RL95-2 were stained with anti-PRL monoclonal Ab. Isotype-matched nonspecific Ab produced no staining (not shown); C. Expression of prolactin receptor in four human carcinoma cell lines. Cells were stained with rabbit polyclonal anti-PRLR Ab. Surface expression of prolactin receptor was measured by flow cytometry. Horizontal bars indicate SE. D. Expression of prolactin receptor in tumors and healthy tissues. Ovarian cancer, endometrial cancer, and endometrial progression (hyperplasia) TMAs were stained with anti-PRLR antibody. Representative cores are presented.

Article Snippet: Mouse monoclonal antibodies against human PRL (catalog no. 53797) and PRLR (catalog no. 53799) were from AnaSpec (San Jose, CA), and Ras inhibitor, α-hydroxy farnesyl phosphonic acid was from Cayman Chemicals (Ann Arbor, MI).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Staining, Produced, Flow Cytometry