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: Nutrients

Article Title: Lactation Activity and Mechanism of Milk-Protein Synthesis by Peptides from Oyster Hydrolysates

doi: 10.3390/nu14091786

Figure Lengend Snippet: Primer sequences for qRT-PCR analysis.

Article Snippet: AKT1, α s1 -casein (CSN1S1) and Cyclin D1 were sourced from Proteintech (Rosemont, IL, USA); P-AKT and p-S6K1 were obtained from Cell Signaling (Danfoss, MA, USA); S6K1, mTOR, P-mTOR, κ-casein (CSN3), β-lactoglobulin (BLG), β-casein (CSN2), prolactin (PRL), prolactin receptor (PRLR), STAT5a, STAT5b and β-actin were obtained from Affinity biosciences, OH, USA; and casein enzymatic hydrolysate was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Techniques:

Journal: Nutrients

Article Title: Lactation Activity and Mechanism of Milk-Protein Synthesis by Peptides from Oyster Hydrolysates

doi: 10.3390/nu14091786

Figure Lengend Snippet: Effects of UEC4 and UEC4-1 on the indices of rats. ( A ) The growth of rats in each group; ( B ) Comparison of body weight gain of offspring rats; ( C ) Mammary gland organ indexes of female rats; ( D ) Serum PRL concentrations of female rats; ( E ) Mammary gland PRL concentrations of female rats; ( F ): Mammary gland PRLR concentrations of female rats. # significantly different from the normal group at p < 0.05, ## significantly different from the normal group at p < 0.01; Δ significantly different from the model group at p < 0.05, ΔΔ significantly different from the model group at p < 0.01; * significantly different from the BXSM group at p < 0.05, ** significantly different from the BXSM group at p < 0.01.

Article Snippet: AKT1, α s1 -casein (CSN1S1) and Cyclin D1 were sourced from Proteintech (Rosemont, IL, USA); P-AKT and p-S6K1 were obtained from Cell Signaling (Danfoss, MA, USA); S6K1, mTOR, P-mTOR, κ-casein (CSN3), β-lactoglobulin (BLG), β-casein (CSN2), prolactin (PRL), prolactin receptor (PRLR), STAT5a, STAT5b and β-actin were obtained from Affinity biosciences, OH, USA; and casein enzymatic hydrolysate was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Techniques: Comparison

Journal: Nutrients

Article Title: Lactation Activity and Mechanism of Milk-Protein Synthesis by Peptides from Oyster Hydrolysates

doi: 10.3390/nu14091786

Figure Lengend Snippet: Effects of UEC4-1 on PRL and PRLR expression. ( A ) The expression of PRL and PRLR in mammary gland; ( B ) The protein levels of PRL and PRLR in mammary gland. * significantly different from the control group at p < 0.05, ** significantly different from the control group at p < 0.01.

Article Snippet: AKT1, α s1 -casein (CSN1S1) and Cyclin D1 were sourced from Proteintech (Rosemont, IL, USA); P-AKT and p-S6K1 were obtained from Cell Signaling (Danfoss, MA, USA); S6K1, mTOR, P-mTOR, κ-casein (CSN3), β-lactoglobulin (BLG), β-casein (CSN2), prolactin (PRL), prolactin receptor (PRLR), STAT5a, STAT5b and β-actin were obtained from Affinity biosciences, OH, USA; and casein enzymatic hydrolysate was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Techniques: Expressing, Control