p stat5 y694  (Danaher Inc)

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) OT1 lymph node cell suspensions were SIINFEKL peptide activated for 36 hr, washed then cultured with no cytokine, IL-15 (20 ng/mL) or IL-2 (20 ng/mL) for 4 or 24 hr. Western blots of PIM1 (two isoforms of 44 and 34 kDa, non-specific band indicated by *), PIM2 (three isoforms of 40, 37, and 34 kDa) or pSTAT5 Y694 expression. ( B ) Schematic of cytokine driven memory and effector CD8 T cell expansion and differentiation cultures. Lymph node or spleen cell suspensions were activated for 2 days with TCR stimulus + cytokine, washed, then split daily into fresh media + cytokine. ( C ) WT (Ly5.1) and Pim dKO LN suspensions were mixed at a 50:50 ratio for T cells and cultured as outlined in ( B ) with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), and CD8 T cell number was measured daily. ( D ) WT and Pim dKO T cells were expanded with IL-15 in separate cultures as per ( B, C ) and % live cells (PI-ve) were assessed on days 4 and 6 (two-way ANOVA). ( E–J ) WT and Pim dKO CD8 T cells were activated with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), expanded with IL-15 as per ( B ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 for parallel RNAseq and proteomic analysis. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( E ) Fold-change in mRNA expression between Pim dKO and WT versus average mRNA expression (TPM). mRNA expression (Transcripts per million, TPM) of ( F ) secondary lymphoid homing receptors Sell, Ccr7, S1pr1 and ( G ) key transcription factors involved in CD8 T cell memory differentiation and maintenance Tcf7, Klf2, Foxo1, Foxo3, Id3. ( H ) WT vs Pim dKO protein copy numbers, differentially expression proteins (FC >1.5, q<0.05) are highlighted in red ( I ) Protein copy numbers per cell for key mitochondrial proteins DRP1, OPA1, and CPT1A. ( J ) WT vs Pim dKO protein copy numbers, mitochondrial proteins (as defined in MitoCarta 3.0) are highlight in pink. Symbols in bar charts represent biological replicates, symbols in ( C, E, H, J ) represent the mean. Error bars show mean ± S.D. Data are representative of ( A ) n=3 or show pooled data from ( C ) n=4, and ( D ) n=5 biological replicates with data collected over at least two independent experiments. Quantitative proteomics and RNAseq was performed on biological triplicates. ** q≤0.01, fold-change (FC) shown on bar graphs when q<0.05. Figure 2—source data 1. PDF files containing labelled and uncropped images for western blots displayed in . Figure 2—source data 2. Original files for western blot images displayed in . Figure 2—source data 3. Raw values plotted in .

Article Snippet: Blots were probed with the following primary antibodies: S6K (5G10, Cell Signaling Technology, cat# 2217 S), S6K p-T389 (108D2, Cell Signaling Technology, cat# 9234 S), STAT5 p-Y694 (C11C5, Cell Signalling Technology, cat# 9359 S), Pim1 (12H8, Santa Cruz, cat# SC-13513), Pim2 (1D12, Santa Cruz, cat# SC-13514).

Techniques: Cell Culture, Western Blot, Expressing, Quantitative Proteomics

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) Estimated copies per cell of PIM1 and PIM2 protein from published quantitative proteomics analysis ; of CD8 T cells expanded in IL-2 or IL-15 as outlined in . ( B–D, F, G ) WT (Ly5.1) and Pim dKO lymph node or spleen single-cell suspensions were mixed at a 50:50 ratio of T cells, activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ). Some of the mixed cell suspensions were also cultured in IL-7 (5 ng/mL) to sustain a naive T cell reference. ( B ) WT and Pim dKO CTL were treated 1 hr +/- Jak1/3 inhibitor Tofacitinib (100 nM; negative control) before pSTAT5 Y694 expression was measured on day 3 and 6 of culture, ( C ) surface CD25 expression was measured on days 3 and 6 of culture, ( D ) CD8 T cell number vs time was calculated, ( F ) CD8 T cell FSC-A, SSC-A and surface activation markers (CD44, CD71) were measured on days 3 and 6 of culture ( G ) expression of adhesion molecule CD62L was measured daily. ( E ) WT and Pim dKO T cells were activated and expanded with IL-2 as per ( B-D ) and ( F, G ) except in separate cultures and % live cells (PI-ve) was assessed on days 4 and 6 (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( D ) represent the mean. Error bars show mean ± S.D. Data are representative of ( B, G ) n=4, ( C, F ) n=6 or show pooled data from ( D ) n=4, ( E ) n=6 biological replicates with data collected over at least two independent experiments. Figure 3—source data 1. Raw values plotted in .

Article Snippet: Blots were probed with the following primary antibodies: S6K (5G10, Cell Signaling Technology, cat# 2217 S), S6K p-T389 (108D2, Cell Signaling Technology, cat# 9234 S), STAT5 p-Y694 (C11C5, Cell Signalling Technology, cat# 9359 S), Pim1 (12H8, Santa Cruz, cat# SC-13513), Pim2 (1D12, Santa Cruz, cat# SC-13514).

Techniques: Quantitative Proteomics, Cell Culture, Negative Control, Expressing, Activation Assay

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: Antibodies targeting p-ERα-S118 (ab32396, Abcam, England), ERα (ab108398, Abcam, England), p-ERK1/2-T202/T204 (4377, CST, USA), ERK1/2 (4695, CST, USA), β-actin (4970, CST, USA), STAT3 (ab68153, Abcam, England), p-STAT3-Y705 (ab76315, Abcam, England), STAT5 (ab16276, Abcam, England), p-STAT5-Y694 (ab32364, Abcam, England), PRLR (ab170935, Abcam, England), rabbit IgG-HRP-linked (7074), mouse IgG-HRP-linked (7076) were used.

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

Journal: Oncology Reports

Article Title: Targeting of STAT5 using the small molecule topotecan hydrochloride suppresses acute myeloid leukemia progression

doi: 10.3892/or.2023.8645

Figure Lengend Snippet: Topotecan hydrochloride specifically binds STAT5. (A) The system specificity of STAT5-FP was tested by binding with STAT family proteins. (B and C) Following incubation of purified STAT5 protein with its labeled STAT5 phosphopeptide [5-carboxyfluorescein GYACHTUNGTRENNUNG(PO3H2)LVLDKW] for 1 h at room temperature, FP signals of two candidate compounds at different drug concentrations were tested and analyzed to identify molecular inhibitory activity. (D) Interaction of topotecan hydrochloride with STAT5. Molecular docking models demonstrated that topotecan hydrochloride bound to multiple amino acids in the SH2 domain of STAT5. (E) The chemical structure of topotecan hydrochloride. (F) Cellular thermal shift analysis, MOLM13 cells were plated and treated with 10 µM of topotecan hydrochloride for 1 h. The samples were put on the PCR instrument and heated at different temperatures for 3 min. After that, liquid nitrogen was used to freeze and thaw samples twice, and cell suspension samples were collected and STAT5 protein levels assessed. FP, fluorescence polarization; KD, dissociation constant.

Article Snippet: The membranes were incubated with antibodies against STAT5 (1;500; cat. no. 25656T; Cell Signaling Technology, Inc.), phosphorylated (p)-STAT5 Y694 (1;500; cat. no. 4322T; Cell Signaling Technology, Inc.), CMYC (1:1,000; cat. no. ab32072; Abcam) and GAPDH (1:1,000; cat. no. ab181602; Abcam) overnight at 4°C.

Techniques: Binding Assay, Incubation, Purification, Labeling, Phospho-proteomics, Activity Assay, Suspension, Fluorescence

Journal: Oncology Reports

Article Title: Targeting of STAT5 using the small molecule topotecan hydrochloride suppresses acute myeloid leukemia progression

doi: 10.3892/or.2023.8645

Figure Lengend Snippet: Topotecan hydrochloride inhibited STAT5 activation in acute myeloid leukemia cells. (A) MOLM13, NB4 and KG1 cells were treated a range of concentrations of topotecan hydrochloride for 24 h. Protein expression levels of p-STAT5, CMYC and STAT5 were assessed using western blotting with GAPDH as a control. (B) MOLM13 cells were treated with topotecan hydrochloride or DMSO (control) for 16 h, and the mRNA expression levels of STAT5 specific target genes were analyzed using reverse transcription-quantitative PCR. (C) Different concentrations of IL-3 and GM-CSF were added to stimulate the cells for 20 min. Then the cells were collected and western blotting was used to assess protein expression levels. (D) Cells were starved for 24 or 48 h in medium without fetal bovine serum and then treated with a range of concentrations of topotecan hydrochloride for 24 h. Cells were then stimulated with 5 ng/ml IL-3 and GM-CSF for 20 min and subjected to western blotting. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. GM-CSF, granulocyte-macrophage colony stimulating factor; ns, not significant; p, phosphorylated.

Article Snippet: The membranes were incubated with antibodies against STAT5 (1;500; cat. no. 25656T; Cell Signaling Technology, Inc.), phosphorylated (p)-STAT5 Y694 (1;500; cat. no. 4322T; Cell Signaling Technology, Inc.), CMYC (1:1,000; cat. no. ab32072; Abcam) and GAPDH (1:1,000; cat. no. ab181602; Abcam) overnight at 4°C.

Techniques: Activation Assay, Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Oncology Reports

Article Title: Targeting of STAT5 using the small molecule topotecan hydrochloride suppresses acute myeloid leukemia progression

doi: 10.3892/or.2023.8645

Figure Lengend Snippet: Topotecan hydrochloride inhibits acute myeloid leukemia tumor growth in vivo . MOLM13 cells were subcutaneously administered at a density of 5×10 6 cells/mouse. After the tumor volume grew to 100–150 mm 3 , the animals were randomly grouped and treated according to group administration. Equal amounts of solvent (n=5), 1 mg/kg/day topotecan hydrochloride (n=5), 3 mg/kg/day topotecan hydrochloride (n=5), or 3 mg/kg/day Azacitidine (n=5) were injected intraperitoneally once a day for 5 days a week at weeks 1, 3 and 4. (A) The tumor volume was calculated as (length × width 2 )/2. (B) After the experiments, all the tumors were weighed. (C) Body weight was measured every three days. IHC analysis was performed on the different treatment groups for p-STAT5, STAT5, Ki-67 and Cleaved Caspase 3. The results were quantified (D) and imaged (E). Scale bar=100 µm. Data are presented as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. TH, topotecan hydrochloride; IHC, immunohistochemistry; p, phosphorylated.

Article Snippet: The membranes were incubated with antibodies against STAT5 (1;500; cat. no. 25656T; Cell Signaling Technology, Inc.), phosphorylated (p)-STAT5 Y694 (1;500; cat. no. 4322T; Cell Signaling Technology, Inc.), CMYC (1:1,000; cat. no. ab32072; Abcam) and GAPDH (1:1,000; cat. no. ab181602; Abcam) overnight at 4°C.

Techniques: In Vivo, Solvent, Injection, Standard Deviation, Immunohistochemistry

Journal: Oncology Reports

Article Title: Targeting of STAT5 using the small molecule topotecan hydrochloride suppresses acute myeloid leukemia progression

doi: 10.3892/or.2023.8645

Figure Lengend Snippet: Topotecan hydrochloride inhibits tumor growth in situ in an acute myeloid leukemia model. MOLM13/Luc cells (1×10 6 ) were injected into the tail vein of pre-irradiated (2.5 Gy) NOD/SCID mice (n=5 per group). The mice were then monitored for seven days and then treated with topotecan hydrochloride or Azacitidine for five weeks. (A) Tumor growth was assessed by weekly bioluminescence imaging. (B) Body weights of mice were measured (n=5). IHC analysis was performed on the different treatment groups for p-STAT5, STAT5, Ki-67, and Cleaved Caspase 3. The results were (C) quantified and (D) imaged. Scale bar=100 µm. Data are presented as mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 ns, not significant; TH, topotecan hydrochloride; IHC, immunohistochemistry; p, phosphorylated.

Article Snippet: The membranes were incubated with antibodies against STAT5 (1;500; cat. no. 25656T; Cell Signaling Technology, Inc.), phosphorylated (p)-STAT5 Y694 (1;500; cat. no. 4322T; Cell Signaling Technology, Inc.), CMYC (1:1,000; cat. no. ab32072; Abcam) and GAPDH (1:1,000; cat. no. ab181602; Abcam) overnight at 4°C.

Techniques: In Situ, Injection, Irradiation, Imaging, Standard Deviation, Immunohistochemistry