Journal: Pharmacological research

Article Title: Ectopic expression of NKG7 enhances CAR-T function and improves the therapeutic efficacy in liquid and solid tumors.

doi: 10.1016/j.phrs.2024.107506

Figure Lengend Snippet: Fig. 4. NKG7 promotes the proliferation of CAR-T cells by enhancing the IL-2 signaling pathway. (A and B) GSEA of DNA replication and cell cycle between B7H3- CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (C) Volcano plot of T cell proliferation-related genes between B7H3-CAR-TNKG7 and B7H3-CAR-T cells. (D) Heatmaps of significant T cell proliferation-related genes. (E and F) IL-2 expression of CAR-T cells was measured by FCM analysis following target cell stimulation for 6 h. (G) Statistics for (E and F). (H) The proliferation of CAR-T cells was detected by a CFSE assay. (I and J) GSEA of the Jak-STAT and PI3K- Akt pathways between B7H3-CAR-TNKG7 and B7H3-CAR-T cells following B7H3 protein stimulation for 6 h. (K) The IL-2 signaling pathway (p-AKT (Ser473), AKT, p- ERK (Thr202/Tyr204), ERK, p-STAT5 (Thr694) and STAT5) in CAR-T cells were explored by WB assays. ***P < 0.001.

Article Snippet: After blocking with 5 % nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies against NKG7 (Cat No. 84835, 1:1000, CST), AKT (Cat No. A11016, 1:1000, ABclonal), p-AKT (Ser473) (Cat No. AP0140, 1:1000, ABclonal), ERK (Cat No. 4695, 1:1000, CST), p-ERK (Thr202/Tyr204) (Cat No. 4370, 1:1000, CST), STAT5 (Cat No. 25656, 1:1000, CST), pSTAT5 (Thr694) (Cat No. 4322, 1:1000, CST) and β-actin (Cat No. 66009–1-Ig, 1:5000, Proteintech) overnight at 4◦C.

Techniques: Expressing, Cell Stimulation, CFSE Assay

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutive activation of JAK2 in the mammary gland increases proliferation during pregnancy and overactivates Stat5 during gestation and lactation. (a) Ki67-positive cells (red) and (b) p-Stat5-positive cells (red) in the mammary glands of JAK2 V617F (control, left) or K14-Cre;JAK2 V617F (right) females at 10 days gestation. E-cadherin staining was used in (a) for visualisation of epithelial cells. Nuclei are in blue. Scale bar: 20 μm. Quantification of Ki67-positive cells (a, n=3 for each genotype) and p-Stat5-positive cells (b, n=5 for each genotype) in ducts and alveoli are shown in the corresponding graphs. (c) Western blot analysis of p-Stat5, p-Stat3 and p-Stat6 in the mammary glands of K14-Cre;JAK2 V617F and control mice (JAK2 V617F and K14 Cre;JAK2 wt) at 24 h lactation (first day post-partum). Total Stats were used for normalisation. Quantification of p-Stat5 signal compared to total Stat5 is shown in (d). Results are mean±S.D. *P<0.05, **P<0.01 versus control mice

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Activation Assay, Staining, Western Blot

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutively active JAK2 in mammary epithelial cells induces Stat5 activation. (a) Representative immunofluorescence staining for JAK2 (red) in KIM-2 cells overexpressing JAK2 wt or JAK2 V617F. MIG (MSCV-IRES-GFP) represents cells carrying the empty vector. Nuclei are in blue. Scale bar: 50 μm. (b) Western blot analysis of JAK2 and the phosphorylation of different Stats in KIM-2 cells harbouring MIG, JAK2 wt or JAK2 V617F. β-Actin and total Stats were used for normalisation. (c) Western blot of JAK2, Stat5 and p-Stat5 in cytosolic and nuclear protein extracts in MIG-, JAK2 wt- and JAK2 V617F-KIM-2 cells. α-tubulin and lamin A/C were used as markers of cytosolic and nuclear fractions, respectively. Experiments were carried out at least twice for two independent sets of retroviral infections

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Activation Assay, Immunofluorescence, Staining, Plasmid Preparation, Western Blot

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutively active JAK2 increases the expression of differentiation markers in mammary epithelial cells in vitro. (a) β-Casein and whey acidic protein (WAP) mRNA expression in cells transiently or stably transduced with empty vector (MIG), JAK2 wt or JAK2 V617F was determined by RT-PCR (representative experiment, n=2). Cyclophilin A was used for normalisation. (b–d) Western blot analysis of β-casein (b), WAP (c), p-Stat5 and Elf5 (d) in KIM-2 cells harbouring MIG, JAK2 wt or JAK2 V617F and incubated with lactogenic hormones for the indicated times (representative images, n=3). β-Actin or total Stat5 were used for normalisation

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Expressing, In Vitro, Stable Transfection, Transduction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutively active JAK2 enhances mammary tumourigenesis. (a) Morphology of KIM-2 cells stably expressing empty vector (MIG), JAK2 wt or JAK2 V617F. Scale bar: 100 μm. (b) Number of colonies in soft agar formed by KIM-2 cells harbouring MIG, JAK2 wt or JAK2 V617F after 3 weeks at the indicated temperatures. Bars represent mean±S.D. of three independent experiments. **P<0.01 versus JAK2 V617F-KIM-2 cells. (c) JAK2 mRNA expression in MCF-7 cells stably transduced with empty vector (MIG), JAK2 wt or JAK2 V617F was determined by RT-PCR (representative experiment, n=2). GAPDH mRNA expression was used for normalisation. (d) Western blot analysis of the phosphorylation of Stat5 in MCF-7 cells harbouring MIG, JAK2 wt or JAK2 V617F (representative images, n=2). Total Stat5 was used for normalisation. (e) Final tumour volume of MIG-, JAK2 wt- and JAK2 V617F-MCF-7-derived xenografts. Mean of each experimental group (n=5) is represented with a horizontal bar and P-values versus JAK2 V617F-MCF-7 xenografts are indicated. (f) Images of MIG-MCF-7- and JAK2 V617F-MCF-7-derived xenografts 6 weeks after the injection of the cells. (g) Representative H&E sections from MIG-MCF-7- and JAK2 V617F-MCF-7-derived xenografts showing well-defined tumour boundary in MIG-MCF-7-derived tumours and invasive clusters of neoplastic cells (marked by an arrow) in JAK2 V617F-MCF-7-derived tumours. Dashed lines indicate tumour margins. Scale bar: 300 μm

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transduction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Injection

Journal: Cells

Article Title: The ADP-Ribosylation Factor 4d Restricts Regulatory T-Cell Induction via Control of IL-2 Availability

doi: 10.3390/cells11172639

Figure Lengend Snippet: Arl4d controls regulatory T-cell conversion. ( A ) Arl4d +/+ or Arl4d −/− splenic CD4 + T cells were differentiated into T H 1, T H 2, or T H 17 cells using a commercial kit. After differentiation, CD4 T cells were stained intracellularly for the signature transcription factors T-bet (T H 1), Gata-3 (T H 2), and Rorγt (T H 17). Additionally, CD4 + T cells were stimulated with PMA and ionomycin and stained intracellularly for the following cytokines: T H 1: IFNγ and TNFα; T H 2: IL-4 and IL-13; T H 17: TNFα and IL-17. Bar graphs show the percentages of transcription-factor- or cytokine-expressing cells within the CD4 + T-cell population. ( B – D ) Arl4d +/+ or Arl4d −/− splenic CD4 + T cells were stimulated with plate-bound αCD3ε (1 μg/mL) in the presence or absence mTGFβ (8 ng/mL) for 48 h, after which cells and supernatants were harvested and analyzed by flow cytometry and ELISA. ( B ) Representative dot plots of T reg induction. Populations shown are gated on viable CD4 + cells. Bar graph also shows the percentage of Foxp3 + cells within the CD4 + T-cell population. ( C ) The percentage of pSTAT5 + cells within the CD4 + T-cell population. ( D ) MFI of pSTAT5 in pSTAT5 + CD4 T cells. Representative data of two ( A ) or three ( B – D ) experiments. Statistical significance was determined by Student’s t test, n.s.: not significant *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: After washing in FACS buffer (PBS, 0.5% FSC, and 0.02% NaN 3 ), cells were permeabilized in 90% ice-cold methanol for 60′. pSTAT5 was stained using an alexafluor647-coupled anti-pSTAT5 antibody (C71E5, Cell Signalling Technologies, Leiden, the Netherlands) in combination with antibodies to CD8α, CD4, CD25, and Foxp3.

Techniques: Staining, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The ADP-Ribosylation Factor 4d Restricts Regulatory T-Cell Induction via Control of IL-2 Availability

doi: 10.3390/cells11172639

Figure Lengend Snippet: Differential capacity for IL-2 production associated with enhanced T reg differentiation induced by Arl4d expression in CD4 T cells. Splenic CD4 + T cells from Arl4d −/− , CD4-Cre + xArl4d Δ/Δ , and Foxp3-Cre + xArl4d Δ/Δ mice and their respective wild-type controls were stimulated with plate-bound αCD3ε (1 μg/mL) in the presence or absence of mTGFβ (8 ng/mL) or 100 IU/mL rhIL-2 for 48 h, after which cells and supernatants were harvested and analyzed by flow cytometry and ELISA. ( A ) Percentages of CD25 + Foxp3 + cells within viable CD4 + T cells and the respective mIL-2 concentration (after αCD3ε stimulation) in the supernatant after 48 h of culture. ( B ) Ex vivo pSTAT5 staining of splenic CD4 + T cells from Arl4d +/+ or Arl4d −/− mice. Dot plots show pSTAT5 staining (black dots) overlayed with isotype staining (blue dots). Bar graphs show the percentages and MFI of pSTAT5 in CD4 + and pSTAT5 + cells, respectively. ( C ) Absolute numbers (#) of Foxp3 + CD4 + cells per μL of blood in Arl4d +/+ and Arl4d −/− mice. ( D ) CD25 high depleted splenic CD4 + T cells were stimulated with plate-bound αCD3ε (1 μg/mL) in the presence or absence of mTGFβ (8 ng/mL) for 48 h, after which cells were stained for CD4, CD25, and Foxp3 ( n = 3). ( E ) Undigested or digested genomic DNA from sorted splenic Arl4d +/+ and Arl4d −/− CD4 + CD25 high T cells was subjected to qPCR analysis of the Foxp3 promoter. Ct values of HpaI-digested samples that lie between the Ct values of undigested and MspII-digested samples indicate partial methylation ( n = 2). Representative data of 3–4 ( A ) and 2 ( B , C ) experiments with n ≥ 3 ( B , C ). Statistical significance was determined by Student’s t test, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, n.s. = not significant.

Article Snippet: After washing in FACS buffer (PBS, 0.5% FSC, and 0.02% NaN 3 ), cells were permeabilized in 90% ice-cold methanol for 60′. pSTAT5 was stained using an alexafluor647-coupled anti-pSTAT5 antibody (C71E5, Cell Signalling Technologies, Leiden, the Netherlands) in combination with antibodies to CD8α, CD4, CD25, and Foxp3.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Ex Vivo, Staining, Methylation

Journal: Cells

Article Title: The ADP-Ribosylation Factor 4d Restricts Regulatory T-Cell Induction via Control of IL-2 Availability

doi: 10.3390/cells11172639

Figure Lengend Snippet: IL-2 receptor signaling and T reg suppressive function in the presence or absence of Arl4d. ( A ) Splenocytes from the indicated mice were isolated and incubated for 10′ with rhIL-2 at the indicated concentrations. Subsequently, cells were fixed and stained for CD8α, CD4, CD25, Foxp3, and pSTAT5 antibodies. The percentages of pSTAT5 + cells were determined in CD8α + T conv , CD4 + T conv , and CD4 + Foxp3 + T reg cells. ( B ) Splenic CD4 + CD25 high T cells were sorted from mice as indicated. These were incubated with CFSE-labeled wild-type CD4 + T cells at the indicated T reg /T eff ratios in the presence of αCD3ε/αCD28-coated beads. After 72 h, the proliferation of the CFSE + CD4 + T cells was determined by flow cytometry. Histograms show an exemplary plot from CD4 + Teff incubated with either CD4 + CD25 high T reg from Arl4d +/+ , Arl4d −/− mice at a 1:1 ratio, or, as a control, CD4 + T eff alone. Bar graphs show the division index. Representative data of 2–3 experiments. ( C ) Percentages and MFI of molecules associated with suppressive function in Arl4d +/+ or Arl4d −/− CD4 + CD25 + Foxp3 + T reg circulating in blood ( n = 5). Statistical significance was determined by Student’s t test, * p ≤ 0.05, # p ≤ 0.01, § p ≤ 0.001, n.s.= not significant.

Article Snippet: After washing in FACS buffer (PBS, 0.5% FSC, and 0.02% NaN 3 ), cells were permeabilized in 90% ice-cold methanol for 60′. pSTAT5 was stained using an alexafluor647-coupled anti-pSTAT5 antibody (C71E5, Cell Signalling Technologies, Leiden, the Netherlands) in combination with antibodies to CD8α, CD4, CD25, and Foxp3.

Techniques: Isolation, Incubation, Staining, Labeling, Flow Cytometry, Control

Journal: Journal of Virology

Article Title: GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination

doi: 10.1128/jvi.01469-23

Figure Lengend Snippet: Polarization of M1 and M2 macrophages in vitro. (A) SDS-PAGE analysis of purified recombinant intact CiIFN-γ2 (17.6 kDa), CiIL-4/13A (17.2 kDa), and CiIL-4/13B (17.1 kDa) proteins. (B and C) Measurement of CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B protein activity by determining the phosphorylation levels of STAT5, ERK, and mTOR. (C) Measurement of phosphorylation levels of STAT5, ERK, and mTOR in three separate experiments by determining the gray value of the WB bands of CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B proteins using the ImageJ software. (D and E) Verification of M1 macrophage polarization by mRNA expression levels of IL-1β, CXCR 3.1, and iNOS (D), as well as enzyme activity of iNOS (E). (F and G) Verification of M2 macrophage polarization by mRNA expression levels of IL-10, CXCR 3.2, and arginase-2 (F), as well as arginase-2 enzyme activity (G). Relative mRNA expression level was normalized to that of EF1a (n = 3) (*P < 0.05 and **P < 0.01).

Article Snippet: Other antibodies used in the study included β-actin mouse mAb (1:10,000, AC004, ABclonal), arginase-2 rabbit pAb (1:500, A6355, ABclonal), iNOS rabbit mAb (1:500, A3774, ABclonal), Bcl-2 rabbit pAb (1:5,000, A0208, ABclonal), HRP rabbit anti-goat IgG (1:5,000, AS029, ABclonal), p-STAT5 rabbit mAb (1:2,000, AP0758, ABclonal), p-ERK rabbit pAb (1:500, AP0472, ABclonal), and p-mTOR rabbit pAb (1:500, AP0094, ABclonal).

Techniques: In Vitro, SDS Page, Purification, Recombinant, Activity Assay, Software, Expressing

Journal: Nature Communications

Article Title: Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK–STAT pathway in Sézary syndrome

doi: 10.1038/ncomms9470

Figure Lengend Snippet: ( a ) Schematic representations of mutations in JAK1, JAK3, STAT3 and STAT5B identified in primary SS samples and the Hut-78 SS-derived cell line. ( b ) Dose–response curves for two primary SS leukaemic cells (SS29 and SS87), the negative control cell lines (HH and Jurkat) and the positive control cell line (Mac-1) after 48 h of treatment with JAK inhibitor I. All experiments were performed in triplicate and error bars represent s.d's (*** P <0.001, T -test). ( c ) Time–response curve for the two primary Sézary syndrome leukaemic cells (SS29 and SS87), the negative control cell lines (HH and Jurkat) and the positive control cell line (Mac-1) treated with 3 μM of JAK inhibitor I. All experiments were performed in triplicate and error bars represent s.d.'s (** P <0.01 and *** P <0.001 T -test). ( d ) Phosphorylation of STAT1, STAT3 and STAT5 in the two primary SS leukaemic cells (SS29 and SS87), the negative control cell lines (HH and Jurkat) and the positive control cell line (Mac-1) treated with 3 μM of JAK inhibitor I for 4 h.

Article Snippet: The following primary antibodies were obtained from Cell Signaling Technology: STAT1 (clone 9H2, 1/1,000), p-STAT1 (clone 58D6, 1/1,000), STAT3 (clone 79D7, 1/1,000), p-STAT3 (clone M9C6, 1/1,000), STAT5 (polyclonal, 1/1,000) and p-STAT5 (clone D47E7, 1/1,000).

Techniques: Derivative Assay, Negative Control, Positive Control, Phospho-proteomics