stat5 inhibitor pimozide  (MedChemExpress)

Journal: Scientific Reports

Article Title: IL-15-Activated CD38 + HLA-DR + CD8 + T cells induce liver injury in cirrhosis via JAK/STAT5 and PI3K/mTOR pathways

doi: 10.1038/s41598-025-02693-6

Figure Lengend Snippet: The signal pathways involved in IL-15-induced innate cytotoxicity of CD38 + HLA-DR + CD8 + T cells. (A) 1 × 10 6 CD8 + T cells from healthy donors were stimulated with IL-15 (20 ng/mL) for 48 h and 72 h, after which phosphorylation of signaling proteins was assessed by flow cytometry for STAT5 ( n = 10), ERK1/2 ( n = 7) and mTOR ( n = 8). Representative dot plots and the summary data show the expression of signaling proteins in CD38 + HLA-DR + CD8 + T cells. (B) The percentage of CD38 + HLA-DR + CD8 + T cells was analyzed after inhibitors treatment ( n = 4). Representative dot plots from a single donor (left) and summary data (right) are presented. (C-G) The percentage NKG2D, FasL, perforin, and Granzyme B in CD38 + HLA-DR + CD8 + T cells were analyzed after inhibitors treatment ( n = 4). (H) CD8 + T cells from healthy donors were pre-treated with STAT5 inhibitor pimozide, MEK inhibitor PD98059, PI3K inhibitor LY294002 or mTOR inhibitor PP242, followed by IL-15 (20 ng/mL) stimulation for the next 72 h. Then, CD8 + T cells were co-cultured with CFSE-labeled K562 cells at a 10:1 E: T ratio and cytotoxicity against K562 cells evaluated ( n = 4). Representative dot plots and the summary data show expression of PI in the gate of CFSE + . (I) Schematic representation of cytokine-mediated crosstalk between T cells and liver cells in liver cirrhosis. The Wilcoxon matched-pairs signed rank test (A) was used for comparisons among groups. The one-way ANOVA (B , C , D-H) was used for comparisons between groups. Control group was indicated treatment only IL-15. ns, not significant, * p < 0.05, ** p < 0.01.

Article Snippet: CD8 + T cells (1 × 10 6 ) from healthy donors were pre-treated with STAT5 inhibitor pimozide, PI3K inhibitor LY294002 or mTOR inhibitor PP242, followed by IL-15 (20 ng/mL) (Thermo, MA, USA) stimulation for the next 72 h. All the inhibitors were obtained from MedChemExpress (NJ, USA).

Techniques: Phospho-proteomics, Flow Cytometry, Expressing, Cell Culture, Labeling, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Plasmalogen-Mediated Activation of GPCR21 Regulates Cytolytic Activity of NK Cells against the Target Cells.

doi: 10.4049/jimmunol.2200183

Figure Lengend Snippet: FIGURE 4. Pls enhance transcriptional activity of STAT5 in NK cells. (A and B) Immunoblotting assays of KHYG-1 cells pretreated with sPls (5 mg/ml) in medium supplemented with 2% FBS for 24 h followed by IL-2 (5 ng/ml) treatments for 20 min. (A) Immunoblotting of p-STAT5 and STAT5 in KHYG-1 cells. The data represent five independent experiments (n 5 5). (B) Quantification data show the relative changes in the ratio of p-STAT5 to STAT5 in (A) (n 5 5). (C) PCR assays show the relative expression of PRF-1 in KHYG-1 cells pretreated with the STAT5 inhibitor pimozide (10 mM for 6 h) followed by sPl (5 mg/ml) treatments for 24 h (n 5 5). (D) Genomic analysis of PRF-1 promoter region. Three pairs of primers for ChIP assays are shown as forward (Fw) and reverse (Rv). (E) ChIP assays show the recruitments of STAT5 transcription factor into the promoter regions of PRF-1 (shown in D) in KHYG-1 cells treated with IL-2 (10 ng/ml) and sPls (5 mg/ml) for 6 h. The data represent three independent experiments (n 5 3). (F) Luciferase assays of the PRF-1 promoter regions in KHYG-1 cells treated with IL-2 (10 ng/ml) and sPls (5 mg/ml) for 6 h (n 5 3). (G) The PRF-1 luciferase promoter (p3: −693, 1100) was transfected in effector KHYG-1 cells followed by recognition of target cancer cells K562 (E:T 5 5:1) in the presence or absence of sPls (5 mg/ml) for 4 h (n 5 5; p < 0.05). The p values (B, C, F, and G) were calculated by ANOVA followed by Bonferroni’s post hoc tests to compare the groups.

Article Snippet: The STAT5 inhibitor (Pimozide) was purchased from R&D Systems.

Techniques: Activity Assay, Western Blot, Expressing, Luciferase, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Plasmalogen-Mediated Activation of GPCR21 Regulates Cytolytic Activity of NK Cells against the Target Cells.

doi: 10.4049/jimmunol.2200183

Figure Lengend Snippet: FIGURE 5. GPCR21 is glycosylated during recognition of the target cancer cells. (A) Schematic diagram showing coculture (recognition) of effector NK cells (KHYG-1) with target cancer cells (K562). It shows the possible effects of GPCR21 to induce Perforin, resulting in apoptosis of the target cancer cells. (B) Immunoblotting assays to detect the protein expression during coculture of KHYG-1 cells and K562 cells at E:T (5:1) for the indicated time periods. The data represent three independent experiments (n 5 3). (C) Two N-linked glycosylation sites (N-x-S/T, where x is any amino acid except proline) in the extra- cellular domain of GPCR21. (D) Immunoblotting data show the effects of sPls treatments (5 mg/ml for 20 min) in the KHYG-1 cells overexpressed with the mutated GPCR21(N to R) constructs. (E and F) PCR assays show the endogenous expression of PRF-1 gene in KHYG-1 cells 48 h after the transfection of the mutated GPCR21 constructs. (F) The relative changes of the expression quantified by ImageJ software (n 5 5). (G and H) Immunoblotting assays show- ing the expression of p-STAT5 protein in KHYG-1 cells overexpressed with the GPCR21 constructs (wild type and mutated). (I) LDH cytotoxicity assays. KHYG-1 cells were transfected with the GPCR21 constructs for 48 h followed by cytolytic assays against K562 cells (E:T 5 10:1) (n 5 5). The values in (F), (H), and (I) represent mean ± SEM, and p values were calculated by ANOVA followed Bonferroni’s post hoc tests.

Article Snippet: The STAT5 inhibitor (Pimozide) was purchased from R&D Systems.

Techniques: Western Blot, Expressing, Glycoproteomics, Construct, Transfection, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Plasmalogen-Mediated Activation of GPCR21 Regulates Cytolytic Activity of NK Cells against the Target Cells.

doi: 10.4049/jimmunol.2200183

Figure Lengend Snippet: FIGURE 6. NK cell activation induces transcription from the GPCR21 promoter. (A) Immunoblotting assays showing the GPCR21 expression after 3-h cocul- ture of NK cells and tumor cells (KHYG-1:K562 5 10:1). (B) ChIP assay shows the recruitment of STAT5 by IL-2 treatments (10 ng/ml for 6 h) on the GPCR21 genomic promoter region upstream of exon 1 in KHYG-1 cells. The data represent three independent experiments (n 5 3). (C) Luciferase activity of the human GPCR21 promoters in KHYG-1 cells treated with IL-2 (10 ng/ml for 4 h) (n 5 4). (D) Luciferase activity from the human GPCR21 promoter con- struct (p −250, 1100) in KHYG-1 cells after recognition of cancer cells (K562) (E:T 5 5:1) (n 5 4; p < 0.01, Student t test). (E) PCR assays (quantification data) show the effects of IL-2 treatments (10 ng/ml for 12 h) in mRNA expression of GPCR21 in KHYG-1 cells pretreated (12 h) with STAT5 inhibitor (pimo- zide, 10 mM) (n 5 3; p < 0.05 and p < 0.01). The p values (C and E) were calculated by ANOVA followed by Bonferroni’s post hoc tests.

Article Snippet: The STAT5 inhibitor (Pimozide) was purchased from R&D Systems.

Techniques: Activation Assay, Western Blot, Expressing, Luciferase, Activity Assay