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

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: T reg cells in VV-resistant tumors have elevated TGFβ and a repressed response to IFNγ. (A) Luminex cytokine analysis of TIF harvested from untreated MEER vvR or MEER vvS implanted in C57BL/6 mice. Three mice per group, three technical repeats per mouse. (B) Active TGFβ 1–3 concentration in the TIF of untreated MEER vvS or MEER vvR tumors as determined by TGFβ reporter assay. (C) TGFβR2 expression on CD8 + , Foxp3− T conv , or Foxp3+ T reg cells in untreated MEER vvR or MEER vvS implanted in Foxp3-reporter mice. Representative histograms from a MEER vvR tumor. 4 repeats, 10 M vvS , 9 M vvR mice. (D) Experimental schema of E–L and Q. Repeated three times. (E–L and Q) Percentage of (E) LAP-TGFβ1+, (F) GARP+, (G) CD103+, (I) TCF1+, (J) CD25 + , (K) CD122+, (L) PD-1+ Tim3+, and percentage and MFI of (H) Nrp1+ and (Q) pSTAT1+ T reg cells by flow cytometry as in D. (M) IFNγ concentration in TIF of MEER vvR and MEER vvS at 7 d after treatment with PBS or VV as in . (N–P) Percentage of pSTAT1 S727 + (N) CD8 + cells, (O) T conv cells, and (P) T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . (R) Representative flow plots and percentage of IFNγ+ T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . Cells were restimulated with PMA/ionomycin direct ex vivo from tumors. M vvS = MEER vvS , M vvR = MEER vvR . Data represent two (A, B, and I–M), three (C, E–H, and Q), or four (N–P and R) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test (A), unpaired T test (B), one-way ANOVA with Sidak’s multiple comparison test paired T test (C, M–P, and R), or paired t test (E–L and Q). ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Luminex, Concentration Assay, Reporter Assay, Expressing, Flow Cytometry, Ex Vivo, Comparison

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: Phenotyping observed in TIL T reg cells is found in dLN and TIL T conv to a lesser extent. (A–I) Quantification of the percentages of dLN T reg cells from paired tumors in Foxp3-Ametrine or Foxp3-RFP mice as in . (A) LAP-TGFβ1+, (B) GARP+, (C) CD103+, (D) Nrp1+, (E) TCF1+, (F) CD25 + , (G) CD122+, (H) PD-1+ Tim3+, and (I) pSTAT1+. (J–Q) Representative flow plots and quantification of T reg phenotyping markers on tumor-infiltrating T conv cells as in . Representative histograms of tumor-infiltrating T conv (Foxp3−) and T reg (Foxp3+) cells are shown for comparison, and quantification axes are scaled for T reg expression. Quantification and flow plots of (J) LAP-TGFβ1+, (K) GARP+, (L) CD103+, (M) Nrp1+, (N) CD25 + , (O) CD122+, (P) Tim3, and PD-1 (Q) pSTAT1+ Tconv cells. (R–U) Representative flow plots and quantification in paired dLN and tumors as in of (R) CD62L and CD44 on T conv cells, (S) CD62L and CD44 on CD8 + cells, (T) TCF1 on T conv cells, and (U) TCF1 on CD8 + cells. CM, central memory CD62L+ CD44 + ; Eff, effector CD62L− CD44 + ; Naïve, CD62L+ CD44 − . Data represent two (R–U) or three (A–Q) independent experiments. Each point represents an individual mouse *P < 0.05, **P < 0.01, ***P < 0.001 by paired T test (A–Q, T, and U) or one way ANOVA with Sidaks multiple comparisons test (R and S). ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Comparison, Expressing

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: TGFβ limits IFNγ signaling and increases T reg cell stability. (A) Immunoblot and densitometry of pSTAT1 Y701 , STAT1, and β-actin in T reg cells sorted from spleen and lymph node of a Foxp3 reporter mouse and treated for 30 min with IFNγ, TGFβ1, or both. (B) Quantification and Cell Trace Violet (CTV) plots of the proliferation of stimulated Thy1.1+ CD4 responder cells in the presence of suppressing T reg cells at the 1:8 T reg cell:responder ratio in an in vitro suppression assay. Percent suppression is normalized to the proliferation index of stimulated CD4 + responder control without T reg cells. T reg cells were sorted from spleen and lymph node of a Foxp3-reporter mouse and then cultured for 3 d in IFNγ, TGFβ, or both. Cells were then sorted again to purify Foxp3+ T reg cells and then co-cultured in the suppression assay with CTV-labeled responder CD4 + cells. (C) Surface Nrp1 expression on sorted T reg cells from spleen and lymph node of a Foxp3-reporter mouse cultured in vitro in varying TGFβ concentrations for 48 h (D–F) An EV control and TGFβ 1 overexpressing (TGFβ OE) line were generated from the MEER vvS line. (D) Tumor growth of C57BL/6 mice implanted intradermally with MEER vvS-EV or MEER vvS-TGFβ OE and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (E) Survival of D. (F) Average tumor growth of MEER vvS-EV and MEER vvS-TGFβ OE as in D. Data represent two (C), four (A and D–F), or five (B) independent experiments; each point or line represents an individual mouse (A–D). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Sidak’s multiple comparison test (A and C), paired T test (B), Mantel-Cox test (E), or mixed effects analysis (F). ns, non-significant. Error bars indicate SEMs. Source data are available for this figure: .

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Western Blot, In Vitro, Suppression Assay, Control, Cell Culture, Labeling, Expressing, Generated, Injection, Comparison

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: VVdnTGFbmm only affects T reg cell phenotype in the tumor. (A) Western blot for TGFβ in EV control and TGFβ 1 overexpressing (TGFβ OE) MEER vvS lines and MEER vvR and MEER vvS as in . (B) Active TGFβ 1–3 levels measured in TIF of LLC, MC38 (colon adenocarcinoma), B16-F10 (melanoma), and C24 ( Pten –/– Braf V600E melanoma) tumors in C57Bl/6 mice as in . The average TGFβ concentration of MEER vvS (light blue) and MEER vvR (gray) from are overlaid as dotted lines. (C) Growth curve of LLC tumors treated with PBS or VV (black arrowhead) as in . (D–N) Representative flow plots and quantification of dLN (D–K) and T reg phenotyping markers on tumor infiltrating T conv cells (L–N) in Foxp3-Ametrine or Foxp3-RFP mice as in . Quantification of dLN (D) percent Foxp3+ of CD4 + and (E) MFI of Foxp3. Quantification and representative flow plots of (F) Nrp1+, (G) pSTAT1+, and (H) LAP-TGFβ1 on dLN T reg cells. Quantification of dLN (I) percent Foxp3− of CD4 + and CD8 + . Quantification and representative flow plots of (J) TNFα and IFNγ in T conv cells with direct ex vivo PMA/ionomycin stimulation and (K) PD-1 and Tim3 on CD8 + cells in dLN. Quantification and representative flow plots of (L) Nrp1+, (M) pSTAT1+, and (N) LAP-TGFβ1 on TIL T conv cells. Data represent two (A–C) or four (D–N) independent experiments. Each dot or line represents a technical repeat (A) or mouse (B–N). *P < 0.05 by one-way ANOVA with Sidak’s multiple comparisons test. ns, non-significant. Source data are available for this figure: .

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Western Blot, Control, Concentration Assay, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: C24 melanoma model has a similar phenotype to MEER vvR after VV treatment. (A) Tumor growth (left, middle) and survival (right) of C57BL/6 mice implanted intradermally with C24 and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (B) Foxp3-Ametrine or Fpxp3-RFP reporter mice implanted intradermally with C24 were as in A. Tumors and lymph nodes were harvested 7 d after treatment for phenotypic analysis. Percentage and total counts of CD8 + T cells and the ratio of T conv cells to T reg cells in treated tumors. (C) Percentage of Foxp3+ CD4 + T conv cells. (D) PD-1 and Tim3 expression on CD8 + cells. (E and F) (E) Production of granzyme B in CD8 + T cells and (F) MFI of pSTAT1 on T conv , CD8 + , and T reg cells as in B. (G) Production of IFNγ in T reg cells as in B after restimulation with PMA and ionomycin as in B. (H) IFNγ measured by ELISA from the TIL of CL24 untreated and 7 d after VV treatment as in B. Mice were implanted with C24 and treated with VV dnTGFβmm and αPD-1 as in . At day 8 after VV treatment, after three doses of αPD-1 were received, tumors were harvested for TIL analysis. (I–N) Representative flow plots and quantifications of (I) IFNγ+ T reg cells, (J) IFNγ + T conv cells, (K) IFNγ + CD8 + cells, (L) Tim3+ PD-1+ T reg cells, (M) TCF1+ T reg cells, and (N) TCF1+ CD8 + cells. Cytokine analysis was performed direct ex vivo with PMA and ionomycin restimulation. (O) Schematic for P and Q. (P) Tumor growth and survival (bottom) of C57BL/6 mice implanted intradermally with bilateral C24 and, when tumors were ∼20 mm 2 , one was treated with a single IT injection of VV dnTGFbmm (injected) at 2.5 × 10 6 PFU/mouse (black arrowhead). Mice were sacrificed when either tumor reached 15 mm in any direction. Starting at 4 d after VV or PBS treatment, mice were given anti-PD-1 or isotype control IP three times a week. (Q) Average growth of H. Data represent two independent experiments. Each point or line represents an individual mouse (A–P). *P < 0.05, **P < 0.01, ****P < 0.0001 by Welch’s T test (B–H), Mantel-Cox test (A and P), unpaired t Test (I–N), or mixed-effects analysis (Q). ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Injection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: Viral delivery of TGFβ inhibition alleviates immunosuppressive T reg cells in resistant tumors. Foxp3-Ametrine or Foxp3-RFP mice implanted intradermally with MEER vvS or MEER vvR were treated with an IT injection of VV ctrl or VV dnTGFβmm at 2.5 × 10 6 PFU/mouse or PBS control. (A–E) Tumors and lymph nodes were harvested 4 (A) or 7 (B–E) d after treatment for phenotypic analysis. (A) Percentage and total counts of T reg cells at day 4 after treatment. (B) Percentage and MFI of Nrp1+ T reg cells at day 7. (C) Percentage of pSTAT1 Ser727 + T reg cells at day 7. (D) MFI of Foxp3 in T reg cells at day 7. (E) Percentage of LAP-TGFβ1+ T reg cells at day 7. (F) Percentage of T conv cells and CD8 + cells 7 d after treatment. (G) Production of TNFα and IFNγ in T conv cells from treated tumors after restimulation with PMA and ionomycin. (H) Percentage of PD-1- and Tim3-expressing CD8 + cells 7 d after treatment. (I) Percentage of TCF1+ in PD-1 and Tim3 CD8 + populations 7 d after treatment, representative plot of PD-1+Tim3− cells. Data represent three (H and I) or four (A–G) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (A–G) or two-way ANOVA (H and I) with Tukey’s multiple comparison test. ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Inhibition, Injection, Control, Expressing, Comparison

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: Journal of Immunotherapy

Article Title: Adoptive Transfer of Interleukin-21-stimulated Human CD8+ T Memory Stem Cells Efficiently Inhibits Tumor Growth

doi: 10.1097/cji.0000000000000229

Figure Lengend Snippet: FIGURE 4. IL-21 only highly upregulates the phosphorylation of STAT3 in the process of TSCM priming. Naive CD8+ T cells display identical STAT phosphorylation profiles in response to IL-2 or IL-21. FACS-purified CD8+ T cells from healthy donors were activated with coated anti-CD3 (2 μg/mL) and soluble anti-CD28(1 μg/mL) overnight, then stimulated with 10 ng/mL IL-2 or 20 ng/mL IL-21. Two hours after stimulation, cells were lysed in sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer. Equivalent amounts of proteins were analyzed by Western blot for indicated phosphoproteins. Data in STAT1 (A) STAT3 (B) STAT5 (C) Akt (D) and Erk1/2 (E) are shown representative of 3 experiments. IL-21 indicates interleukin-21; STAT, signal transducer and activator of transcription; TSCM, T memory stem cell.

Article Snippet: Total protein (20 μg) was separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel followed by standard immunoblotting with primary antibodies for Stat1, phospho-Stat1 (Tyr701), Stat3, phospho-Stat3 (Tyr705, Ser727), Stat5, phospho-Stat5 (Tyr694), Akt, phospho-Akt (Ser473), Erk1/2, phospho-Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA), and GAPDH (Proteintech, Wuhan, China) or α-tubulin (Cell Signaling Technology), followed by fluorescence-conjugated goat anti-mouse or antirabbit IgG (LI-COR, Lincoln).

Techniques: Phospho-proteomics, Purification, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Journal of Neuroinflammation

Article Title: The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

doi: 10.1186/1742-2094-9-113

Figure Lengend Snippet: Effects of SAHA on IFN-γ-induced phosphorylation of STAT1 and STAT3 in human astrocytes. Human astrocytes were incubated with or without SAHA at the concentrations indicated for 1 h. The cells were subsequently stimulated with IFN-γ for 30 minutes. Astrocytes in the control group were incubated with medium only. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr 701 -STAT1 (pSTAT1) and total STAT1 (A) or phospho-Tyr 705 -STAT3 (pSTAT3) and total STAT3 (C) . The density ratios of phosphorylated to total protein are shown as mean ± S.E.M. of three independent experiments (B, D) . *Significantly different from IFN-γ stimulation only.

Article Snippet: The protein was transferred to a PVDF membrane at 70 V for 2 h. The membrane was blocked with 5% skim milk plus 3% bovine serum albumin (BSA) in PBS at room temperature (RT) for 1 h. Subsequently, the membrane was incubated with specific rabbit antibodies against phospho-Tyr 701 -STAT1 (1:2,000), total STAT1 (1:1,000), phospho-Tyr 705 -STAT3 (1:2,000) or total STAT3 (1:1,000) at 4°C overnight and then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000) at RT for 1 h. All antibodies used for immunoblotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Incubation, Control, SDS Page

Journal: Cell Reports Medicine

Article Title: Co-targeting RANK pathway treats and prevents acquired resistance to CDK4/6 inhibitors in luminal breast cancer

doi: 10.1016/j.xcrm.2023.101120

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Article Snippet: Rabbit monoclonal anti phospho-STAT1 (Tyr701) (D4A7) , Cell Signaling , Cat#7649; RRID:AB_10950970.

Techniques: Recombinant, Control, shRNA, Blocking Assay, Alamar Blue Assay, Flow Cytometry, Isolation, cDNA Synthesis, Bicinchoninic Acid Protein Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Western Blot, Software

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces STAT1 activation in BV2 cells. BV2 cells were exposed to 20 ng/mL of IFNγ at different time points and activation of STAT1 was evaluated through different experiments. ( A ) Total protein extracts of IFNγ-treated BV2 cells at different time points were analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody, and after stripping, the same blot was analyzed with anti-STAT1 antibody. Densitometric analysis revealed a STAT1 phosphorylation upon IFNγ treatment, peaking at 15 min compared with untreated BV2 cells (CTR). ( B ) RT-qPCR of iNOS and COX2, two STAT1-dependent genes, revealed an increase in their mRNA levels after 18 and 24 h of IFNγ treatment in BV2 cells. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. untreated cells (CTR). Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Stripping Membranes, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment counteracts IFNγ-dependent STAT1 activation in BV2 cells. ( A ) Total protein extracts from BV2 cells pre-treated overnight with GEE and subsequently exposed to 20 ng/mL IFNγ for 18 h were analyzed by Western Blot using a phospho-Tyr701 STAT1 antibody. The same blot was analyzed with anti-Tubulin antibody to check the amount of loaded proteins. Densitometric analysis showed decreased STAT1 phosphorylation in GEE pre-treated cells compared to CTR. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). # p < 0.05 vs. IFNγ-treated cells. ( B ) RT-qPCR analysis revealed that iNOS and COX2 mRNA levels were reduced in BV2 cells pre-treated overnight with GEE and then exposed to 20 ng/mL IFNγ for 24 h. Data are expressed as mean ± SD (n = 3). ** p < 0.01 vs. CTR, ## p < 0.01 vs. IFNγ-treated cells. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment inhibits IFNγ-induced STAT1 phosphorylation and nuclear translocation in BV2 cells. The cells were immunostained with phospho-Tyr701 STAT1 (red) and analyzed by confocal microscopy (lens 60×). Nuclei were stained with DAPI (cyan). The orthogonal sections (ZX and ZY) of merge image reveal that pTYR 701 STAT1 protein is inside the nuclei. Scale bars indicate 10 μm. Images are representative of three separate experiments.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Translocation Assay, Confocal Microscopy, Staining

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces S-glutathionylation of STAT1. ( A ) Total protein extracts of BV2 cells left untreated (CTR) or treated with 20 ng/mL IFNγ for the indicated times were subjected to immunoprecipitation with anti-STAT1 antibody. Immunoprecipitated STAT1 (IP: STAT1) was analyzed by Western Blot under non-reducing conditions using anti-SSG antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Densitometric analysis revealed increased STAT1 S-glutathionylation after 5 and 15 min of IFNγ treatment compared to untreated BV2 cells. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). ( B ) Total protein lysates from the same untreated and treated BV2 cells were reserved before pull-down (input) and analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Images are representative of four independent experiments. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Immunoprecipitation, Western Blot, Membrane, Stripping Membranes

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: FNγ induces M1 polarization in BV2 cells through oxidative stress and STAT1 activation. ( A ) The expression of the pro-inflammatory cytokines IL-6 and TNFα in the supernatant of BV2 cells was evaluated by ELISA after 18 h of IFNγ stimulation, with or without GEE pretreatment. GEE markedly reduced the IFNγ-induced secretion of both cytokines, confirming the involvement of oxidative stress in M1 polarization. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ. ( B ) Parental and STAT1 knockdown in BV2 cells were treated with 20 ng/mL of IFNγ for 18 h. Total protein extracts were analyzed by Western Blot with anti-iNOS and anti-Actin antibodies. Densitometric analysis revealed reduced iNOS induction in STAT1 knockdown in BV2 cells compared with parental cells upon IFNγ treatment. Parental and STAT1 knockdown in BV2 cells cultured without IFNγ were used as controls (CTR). Images are representative of three independent experiments. Single data points are shown as red dots. * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Cell Culture