phosphorylated stat5  (Cell Signaling Technology Inc)

Journal: Animal Nutrition

Article Title: Myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα) mediates methionine- and leucine-stimulated β-casein synthesis in bovine mammary epithelial cells via targeting mTOR

doi: 10.1016/j.aninu.2025.01.003

Figure Lengend Snippet: Silencing of MRCKα inhibits the stimulatory effects of Met and Leu on mRNA abundance of genes related to milk protein synthesis in bovine mammary epithelial cells. Cells were transfected with scrambled siRNA (control, SC) or MRCKα siRNA (SI) and stimulated simultaneously with control (B, basal medium), additional Met (M, Met = 0.6 mM) or Leu (L, Leu = 0.6 mM). After 24 h, cells were collected and isolated for RT-qPCR analysis. Values are presented as mean ± SEM for three independent experiments. P AA , P -value of amino acid addition (AA); P SI , P -value of silencing of MRCKα (SI); P AA×SI , P -value of AA × SI interaction. CSN2 = β-casein; CSN1S1 = αS1-casein; CSN3 = κ-casein; LALBA = lactalbumin α; MRCKα = myotonic dystrophy-related CDC42-binding kinase alpha; mTOR = mechanistic target of rapamycin; JAK2 = Janus kinase 2; STAT5 = signal transducers and activators of transcription 5.

Article Snippet: Rabbit polyclonal anti-human signal transducer and activator of transcription 5 (STAT5) antibody, rabbit polyclonal anti-human phosphorylated STAT5 (Tyr 694 ) antibody, rabbit polyclonal anti-human PI3K antibody and rabbit polyclonal anti-human phosphorylated PI3K (Tyr 317 ) antibody were purchased from Bioss antibodies (Beijing, China).

Techniques: Transfection, Control, Isolation, Quantitative RT-PCR, Binding Assay

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