Journal: Acta Pharmaceutica Sinica. B

Article Title: Macrophage P2Y 6 R activation aggravates psoriatic inflammation through IL-27-mediated Th1 responses

doi: 10.1016/j.apsb.2024.06.008

Figure Lengend Snippet: Macrophage P2Y 6 R mediated Th1 type psoriasis-like inflammation via p-JAK2/p-STAT1/T-bet signaling. (A) Flow diagram. (B, C) FACs and the statistical analysis of IFN γ + CD4 + T-cells ( n = 3). (D) IFN γ concentration after WT or P2Y 6 R −/− mice BMDM supernatant inducing Naïve CD4 + T-cells ( n = 4). (E) The mRNA expression of the Th1-associated cytokines, Ifnγ , Cxcl9 , Cxcl10, and Cxcl11 were detected by RT-qPCR ( n = 3). All mRNA levels were normalized to GAPDH. (F) Linear regression analysis indicated a positive correlation between P2ry6 and Stat1 in GSE181318 and GSE14905. (G) GSEA analysis of GSE50400 showed JNK/STAT signaling pathways were up-regulated in IMQ-induced mice. (H) Top 15 putative transcription factors based on ChEA3. (I, J) The protein expression levels of p-JAK2, p-STAT1, and T-bet in T cells. Normal protein levels were normalized to β -actin, the values for the phosphorylated forms of proteins were normalized to phosphorylation-independent levels of the same protein ( n = 4). (K) CD4 + Naïve T cells supernatant IFN γ concentration after WT or P2Y 6 R −/− mice BMDM supernatant inducing and being treated with or without RO8191 ( n = 4). (L) The proportion of IFN γ + CD4 + T cells of CD4 + Naïve T cells after WT or P2Y 6 R −/− mice BMDM supernatant inducing and being treated with or without RO8191 ( n = 3). (M, N) T-bet protein expression of CD4 + Naïve T cells after WT or P2Y 6 R −/− mice BMDM supernatant inducing and being treated with or without RO8191 ( n = 4). Data are expressed as mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The following antibodies were used: PLC β (bs-6976R, Bioss, Beijing, China), PKC (ab181558, Abcam, Cambridge, UK), p-PKC (EP2730Y, Abcam), p38 (AF6456, Affinity, Jiangsu, China), p-p38 (AF4001, Affinity), ERK (bs-0022R, Bioss), p-ERK (bs-3016R, Bioss), p-JAK2 (A19629, Abclonal, Wuhan, China), JAK2 (AP0531, Abclonal), p-STAT1 (bs1657R, Bioss), STAT1 (10144-2-AP, Proteintech, Wuhan, China), p-JNK (bs1640R, Bioss), JNK (AF6318, Affinity), T-bet (14-5825-82, Abclonal), β -actin (T0022, Affinity).

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Characterisation of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC-βH5 beta cells

doi: 10.1101/2022.11.29.518315

Figure Lengend Snippet: (a) Immunoblotting of cleaved caspase 7 following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNα and 1000 U/mL TNFα) (Mix) for 96 h. Tubulin was used as loading control (n=3). (b) Immunoblotting of IκBα, phosphorylated STAT1, and phosphorylated JNK following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNγ and 1000 U/mL TNFα) alone or in combination (Mix) for 30 min. GAPDH was used as loading control. Representative image (n=3). Lysates from INS-1E cells with/without cytokine exposure were used as positive control. Gene expression of (c) DDIT3/CHOP, (d) HRK/DP5 and (e) CXCL10 in cells exposed to individual cytokines for 48 h. GAPDH was used as housekeeping gene. Data are means ± SEM (n=4), *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Antibodies used were anti-cleaved caspase-7 (#9491S, Cell Signaling, 1:500), anti-MHC-I (#ALX-805-711-C100, Enzo, Farmingdale, NY, USA, 1:2000), anti-phosho-c-Jun N-terminal kinase (P-JNK) (#9252, Cell Signaling, 1:1000), anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) (#J1512, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), anti-phospho-signal transducer and activator of transcription 1 (P-STAT1) (7649S, Cell Signaling, 1:1000), anti-Tubulin (T8203, Sigma-Aldrich, St. Louis, MO, USA, 1:2000), anti-GAPDH (#9482, Abcam, Cambridge, UK, 1:5000) and secondary HRP-conjugated anti-mouse (#7076) or anti-rabbit (#7074) (Cell Signaling) IgG antibodies.

Techniques: Western Blot, Control, Positive Control, Gene Expression

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: ( A ) Spleen leukocytes were stimulated with PHA for 12 h. Relative mRNA levels of T-bet, STAT1 and STAT4 were examined by qPCR, n = 4. ( B ) Flow cytometry showed the phosphorylation level of STAT1 in lymphocytes that stimulated with PHA. ( C ) Transcriptional levels of IFN-γ in HEK 293T cells that transfected with tilapia T-bet, STAT1 and STAT4, n = 6. ( D ) HEK 293T cells were co-transfected with tilapia T-bet, STAT1 or STAT4, and pGL3-IFN-γ promoter. The LUC activities were assessed at 48 h post-transfection, n = 4. ( E-G ) Spleen leukocytes from tilapia that i . p . injected with T-bet-specific, STAT1-specific or control siRNA for 2 days were harvested and stimulated with CD3ε mAb for 12 h. Relative mRNA levels of STAT1, T-bet and IFN-γ ( E, F , n = 4), and the percentage of CD3 + CD4-1 + IFN-γ + T cells ( G ) were examined. ( H-L ) Tilapia i . p . injected with T-bet-specific, STAT1-specific or control siRNA were infected with E . piscicida . Tilapia was i . p . injected with BFA 6 h before sacrifice, and spleen leukocytes were harvest for assay. Western blot assay showed the expression of IFN-γ at 48 h post infection ( H ). Flow cytometry and scatter plot figures showed the percentage and absolute numbers of CD3 + CD4-1 + T cells (I, K) and CD3 + CD4-1 + IFN-γ + T cells (J, L) on 5 DPI, n = 4. ( M-O ) Tilapia individuals that infected with E . piscicida were injected with STAT1 inhibitor Fludarabine or PBS, and animals were i . p . injected with BFA 6 hours before sacrifice. ( M ) Western blot assay showed the protein levels of IFN-γ in spleen leukocytes on indicated days. ( N ) Flow cytometry showed the percentage of IFN-γ in spleen CD4-1 + T cells on day 7 post-infection. ( O ) Kaplan-Meyer survival plot showed the survival percentage of tilapia, n = 25. These experiments were repeated for at least two independent times. *: p <0.05, **: p <0.01, ***: p <0.001, determined by a two-tailed Student’s t-test. The accession numbers of selected sequences were listed in .

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: Flow Cytometry, Phospho-proteomics, Transfection, Injection, Control, Infection, Western Blot, Expressing, Two Tailed Test

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: ( A, B ) Spleen leukocytes were stimulated with PHA or CD3ε/CD28 mAbs for 12 h, and mRNA levels of IFNγR1 and IFNγR2 were examined by qPCR, n = 6. ( C ) SDS-PAGE assay showed the recombination of tilapia IFNγR1 and IFNγR2 with GST-tag in E . coli . ( D ) GST pull-down assay showed the interaction of tilapia IFN-γ with IFNγR1 and IFNγR2. ( E ) Flow cytometry showed the phosphorylation level of STAT1 in lymphocytes that stimulated with recombinant IFN-γ. ( F, G ) Spleen leukocytes were stimulated with recombinant IFN-γ for 12 h, and mRNA levels of indicated molecules were examined by qPCR, n = 5. ( H ) Tilapia was injected with STAT1 inhibitor for 2 days before spleen leukocytes were stimulated with recombinant IFN-γ for 12 h. The expression levels of T-bet were examined by qPCR, n = 6. ( I-K ) Spleen leukocytes from tilapia that i . p . injected with T-bet-specific, STAT1-specific or control siRNA for 2 days were harvested and stimulated with recombinant IFN-γ for 12 h. Relative mRNA levels of STAT1, T-bet and IFN-γ ( I, J , n = 5), and the percentage of IFN-γ + cells in gated CD3 + CD4-1 + T cells ( K ) were examined. These experiments were repeated for at least two independent times. *: p <0.05, **: p <0.01, ***: p <0.001, determined by a two-tailed Student’s t-test.

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: SDS Page, Pull Down Assay, Flow Cytometry, Phospho-proteomics, Recombinant, Injection, Expressing, Control, Two Tailed Test

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: IL-2-mTORC1 signaling coordinates STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in tilapia.

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: Cell Differentiation

Journal:

Article Title: Signaling of short- and long-term regulation of intestinal epithelial type 1 Na + /H + exchanger by interferon- γ

doi: 10.1038/sj.bjp.0706167

Figure Lengend Snippet: Abundance of total STAT1, phospho-STAT1 and phospho-p38 MAPK in Caco-2 cells treated with vehicle and IFN-γ (1000 U ml−1) or anisomycin (100 nM) for 1, 3 and 24 h.

Article Snippet: Total and phosphorylated STAT1 protein levels in Caco-2 cells exposed to IFN- γ (1000 U ml −1 ) and anisomycin (100 n M ) for 1, 3 and 24 h were analyzed using the PhosphoPlus® Stat1 (Tyr701) antibody kit (Cell Signaling TechnologyTM) according to the manufacturer's protocol.

Techniques:

Journal:

Article Title: Signaling of short- and long-term regulation of intestinal epithelial type 1 Na + /H + exchanger by interferon- γ

doi: 10.1038/sj.bjp.0706167

Figure Lengend Snippet: Effect of EGCG (20 μM), SB 203580 (10 μM) and PKC downregulation (PKC dr; overnight treatment with 100 nM PDBu) on the abundance of STAT1 and phospho-STAT1 in Caco-2 cells treated for 24 h in the absence (vehicle) and presence of IFN-γ (1000 U ml−1).

Article Snippet: Total and phosphorylated STAT1 protein levels in Caco-2 cells exposed to IFN- γ (1000 U ml −1 ) and anisomycin (100 n M ) for 1, 3 and 24 h were analyzed using the PhosphoPlus® Stat1 (Tyr701) antibody kit (Cell Signaling TechnologyTM) according to the manufacturer's protocol.

Techniques:

Journal: Nature immunology

Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

doi: 10.1038/s41590-019-0480-4

Figure Lengend Snippet: (a-c) Quantification of (a) Slamf6 + Tim-3 – , (b) Slamf6 + Tim-3 + , and (c) Slamf6 – Tim-3 + subsets following in vitro stimulation (αCD3/CD28) of control or Ptpn2 -deleted naive CD8 + T cells in the presence of indicated cytokines or blocking antibodies. Representative of two pooled experiments, n ≥ 4 technical replicates. (d) Quantification of pSTAT1 expression of splenic CD8 + T cells day 6 post LCMV Clone 13 infection for co-transferred control and Ptpn2 -deleted cells following ex vivo restimulation with IFN-α. Representative of two independent experiments, n = 5 biological replicates. (e-f) Quantification of pSTAT1 in (e) Slamf6 + or (f) Tim-3 + cells following ex vivo IFN-α restimulation of co-transferred control and Ptpn2 -deleted cells as in (d). Representative of two independent experiments, n = 5 biological replicates. (g) Quantification of frequencies of co-transferred control and Ptpn2 -deleted CD8 + T cells day 4 post LCMV Clone 13 infection following treatment with isotype (left graph) or IFNAR blocking antibody (right graph). Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 3 biological replicates. (h) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFNAR blocking antibody (right). Representative of two independent experiments, n = 5 biological replicates. (i) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFN-γ neutralizing antibody (right). Representative of two independent experiments, n = 5 biological replicates. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-way ANOVA (a-c) or two-sided Student’s paired t-test (d-i) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

Article Snippet: Antibodies and dyes were purchased from BD Biosciences (7-AAD Cat# 559925 (1:100 dilution), Slamf6 Clone 13G3 Cat# 561540 (1:100 dilution), BrdU Clone 3D4 Cat# 552598 (1:50 dilution), Ki67-PerCP-Cy5.5 Clone B56 Cat# 561284 (1:100 dilution)); Biolegend (B220 Clone RA3–6B2 Cat# 103208, 103226 (1:100 dilution), CD11b Clone M1/70 Cat# 101208, 101216 (1:100 dilution), CD127 Clone A7R34 Cat# 135014, 135024 (1:100 dilution), CD25 Clone PC61 Cat# 101904 (1:100 dilution), CD3ε Clone 17A2 Cat# 100220, 100308, 100336, 100328 (1:100 dilution), CD4 Clone RM4–5 Cat# 100516, 100531, 100543 (1:100 dilution), CD44 Clone IM7 Cat# 103008, 103028, 103030 (1:100 dilution), CD45.1 Clone A20 Cat# 110708, 110716, 110741 (1:100 dilution), CD45.2 Clone 104 Cat# 109824, 109832, 109830 (1:100 dilution), CD5 Clone 53–7.3 Cat# 100608 (1:100 dilution), CD62L Clone MEL-14 Cat# 104417 (1:100 dilution), CD8α Clone 53–6.7 Cat# 100737 (1:100 dilution), CD8β Clone YTS156.7.7 Cat# 126606, 126608, 126610, 126620, 126614 (1:100 dilution), c-Kit Clone ACK2 Cat# 135108 (1:100 dilution), CXCR5 Clone L138D7 Cat# 145509 (1:50 dilution), Gr-1 Clone RB6–8C5 Cat# 108408 (1:100 dilution), Granzyme B Clone GB11 Cat# 515403, 515406 (1:100 dilution), I-A/I-E Clone M5/114.15.2 Cat# 107614 (1:100 dilution), IFN-γ Clone XMG1.2 Cat# 505810 (1:100 dilution), IFNAR1 Clone MAR1–5A3 Cat# 127314 (1:100 dilution), Ly-6c Clone HK1.4 Cat# 128007 (1:100 dilution), PD-1 Clone 29F.1A12 Cat# 135206, 135209 (1:100 dilution), Sca-1 Clone D7 Cat# 108108, 108128 (1:100 dilution), TCR Vα2 Clone B20.1 Cat# 127814, 127806 (1:100 dilution), TCR Vβ5 Clone MR9–4 Cat# 139506 (1:100 dilution), TCR Vβ8 Clone KJ16–133.18 Cat# 118406 (1:100 dilution), TER-119 Clone TER-119 Cat# 116208 (1:100 dilution), TCF1 Clone 7F11A10 Cat# 655208 (1:100 dilution), Tim-3 Clone RMT3–23 Cat# 119703, 119723 (1:100 dilution), TNF Clone MP6-XT22 Cat# 506322 (1:100 dilution), TruStain fcX Clone 93 Cat# 101320 (1:50 dilution), Rat IgG2a κ Isotype Clone RTK2758 Cat# 400508 (1:100 dilution), Rat IgG2b κ Isotype Clone RTK4530 Cat# 400612 (1:100 dilution), Streptavidin Cat# 405225 (1:400)); Invitrogen anti-GFP (Polyclonal) Cat#A21311 (1:350 dilution); Thermo Fisher Scientific (Foxp3 Clone FJK-16s Cat# 48-5773-82 (1:50 dilution), Near-IR Fixable Live/Dead Cat# L34976 (1:500 dilution)); and Cell Signaling Technology pSTAT1 Clone 58D6 Cat# 9174 (1:100 dilution).

Techniques: In Vitro, Blocking Assay, Expressing, Infection, Ex Vivo, Standard Deviation