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: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay