Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Dietary Lauric Acid Suppresses Inflammation, Cholestasis, Hepatocyte Injury, and Senescence in 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine-induced Inflammatory Cholangiopathy

doi: 10.1016/j.jcmgh.2026.101731

Figure Lengend Snippet: Effect of lauric acid on expression of inflammation, senescence, and oxidative stress genes and biomarkers in hepatocytes isolated from experimental mouse groups. qRT-PCR-mRNA analysis in hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( A ) Genes involved in inflammation and oxidative stress. ( B ) Senescence genes. ( C ) Mitochondrial fatty acid oxidation and oxidative stress genes. ( D ) β-gal staining ( yellow arrow ) as a senescence marker in overnight cultured primary hepatocytes isolated from chow, DDC, LA, and DDC/ LA mice. Bar graph showing the number of senescent (β-gal + ) cells per microscopic field in liver sections from chow, DDC, DDC/LA, and LA groups. ( E ) Immunoblotting and quantification of β-gal in fresh hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( F ) Serum hydrogen peroxide levels and CCL2 concentrations (ELISA), and lipid peroxides, nitrite, and malondialdehyde levels from isolated hepatocytes. ( G–H ) Co-culture of human cholangiocytes H69 ( upper wells ) and Huh7 cells was conducted. H69 cells were incubated with and without DDC overnight in the presence or absence of DLPC. ( G ) mRNA expression by qPCR was measured in Huh7 cells ( bottom wells ) for CCL2, CDKN1A, NR0B2, and ABCB11 and ( H ) in H69 cells for CCL2, CDKN1A, KRT19, CTGF, and TNF . ( I ) Primary mouse hepatocytes were incubated with CCL2 in the presence or absence of DLPC overnight, and mRNA expression by qPCR was analyzed for senescence genes Cdkn1a and Cdkn1b . ( J ) β-gal staining ( yellow arrow = senescent cells) of cultured Huh7 cells incubated with CCL2 overnight in the presence or absence of DLPC. Bar graph showing the number of senescent (β-gal + ) Huh7 cells after treatment with CCL2 alone or in combination with DLPC. CCL2 treatment markedly increased cellular senescence, which was inhibited by DLPC. ( K ) Confocal fluorescence microscopy of primary cultured hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P16 ( red ), and nuclear DAPI ( blue ) from showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. ( L ) Confocal fluorescence microscopy of primary mouse hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P21/WAF/CIP ( red ) and nuclear DAPI ( blue ) also showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. For ( G–I ), data points represent replicates in 3 independent experiments. For ( D and J ), photomicrographs are shown that are representative of 3 separate experiments. For ( K and L ), immunofluorescent images are shown that are representative of 3 separate experiments. ( M ) Western analysis of pSTAT1 protein, total STAT1, and actin expression in hepatocytes isolated from the mouse groups, including quantification of integrated density values (IDVs). ( N ) ChIP assay of hepatocytes isolated from mouse groups for STAT1 binding to the promoter region of Cdkn1b using a STAT1-specific antibody. ( O ) Western analysis of pSTAT1 protein expression in cultured primary mouse hepatocytes exposed to CCL2 overnight in the presence or absence of DLPC, including quantification of IDVs of immunoblots. Statistical analysis was performed by 1-way ANOVA with Tukey’s correction for multiple comparisons. a P < .05 vs all other groups; b P < .05 vs DDC; b P < .05 vs CCL2.

Article Snippet: Phospho-STAT1 , 9167 , 1:1000 , Cell Signaling.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Staining, Marker, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Statins act as transient type I interferon inhibitors to enable the antitumor activity of modified vaccinia Ankara viral vectors

doi: 10.1136/jitc-2020-001587

Figure Lengend Snippet: Simvastatin and atorvastatin reduce surface expression of IFNAR1 and protein endocytosis. (A) L929 cells were incubated with different concentrations (from 50 µM until 6.5 µM) of simvastatin for 3 hours, and levels of IFNAR1 on the cell membrane were quantified by flow cytometry. Normalized GM: the normalized geometric mean was calculated considering as 100% the geometric mean of the cells stained with the anti-IFNAR1 mAb and as 0% the geometric mean of the cells with isotype control. N=3. (B) Representative histogram of mock-treated cells without αIFNAR1 staining (black dashed line), simvastatin treated cells stained with αIFNAR1-PE (red line) and mock-treated cells stained with αIFNAR1-PE (black line). (C) As in a but for compound atorvastatin, in this case two fold dilution curve was made from 600 µM. N=3. (D) Representative histogram of mock-treated cells without αIFNAR1 staining (black dashed line), simvastatin treated cells stained with αIFNAR1-PE (blue line) and mock-treated cells stained with αIFNAR1-PE (black line). (E) L929 cells were incubated with 37.5 µM simvastatin or 300 µM atorvastatin for 3 hours, and BSA-FITC internalization was quantified by flow cytometry. N=3. One-way ANOVA followed by Sidak’s post-test. ***P<0.001. (F) Representative histogram of simvastatin treated cells (red line), atorvastatin treated cells (blue line) and mock-treated cells (black line). ANOVA, analysis of variance; BSA-FITC, bovine serum albumin-fluorescein 5 (6)-isothiocyanate; IFNAR1, interferon receptor.

Article Snippet: Later, the supernatant was eliminated and IFNα alone (3000 units/ml) was added for 30 min and then Western blotting was accomplished using a rabbit polyclonal antibody to β-Actin (A2066, Sigma, Stockholm, Sweden), a rabbit monoclonal antibody to phopho-STAT1 (Tyr701) (58D6, Cell Signaling Technology, Danvers, MA) and a rabbit polyclonal antibody to STAT1 (ref. 9172, Cell Signaling Technology, Danvers, Massachusetts, USA).

Techniques: Expressing, Incubation, Membrane, Flow Cytometry, Staining, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Statins act as transient type I interferon inhibitors to enable the antitumor activity of modified vaccinia Ankara viral vectors

doi: 10.1136/jitc-2020-001587

Figure Lengend Snippet: CD8 + T lymphocytes are required for the antitumor activity of MVA-OVA combined with simvastatin, whereas CD4 + T lymphocytes and NK cells are detrimental. B16-OVA melanoma cells were injected subcutaneously. Seven and fourteen days later, mice were treated intraperitoneally with vehicle or simvastatin (20 µg/mice), after 2 hours, MVA-OVA (5×10 7 TCID 50 per mouse) was administered subcutaneously in the tumor area. Anti-CD8 mAb, anti-CD4 mAb or anti-NK1.1 (200 µg/mice) were administered intra-peritoneally 1 day before first therapeutic treatment administration and on days +2, +6, +9 and +13. Anti-CD25 (250 µg/mice) was administered intra-peritoneally 1 day before first therapeutic treatment administration. (A) Individual follow-up of mean tumor diameters indicating the fraction of mice completely rejecting established tumors. N=5 and 6 in the group treated with anti-CD25 (B) Mean+SEM of the different experimental groups. data was fitted to a third order polynomial and compared using extra sum-of-squares F test with Bonferroni correction. ***p<0.0001. (C) Overall survival of the indicated treatment groups. Log-rank test with Benjamini-Hochberg correction. **p<0.01. (D) B16-OVA tumor-bearing mice were treated as described above and 15 days after, immune infiltration in the tumor microenvironment was analyzed by flow cytometry. N=6. One-way ANOVA followed by Sidak’s post-test. No significant differences were detected. ANOVA, analysis of variance; B16-OVA; B16 melanoma expressing ovalbumin; MVA, modified vaccinia virus Ankara.

Article Snippet: Later, the supernatant was eliminated and IFNα alone (3000 units/ml) was added for 30 min and then Western blotting was accomplished using a rabbit polyclonal antibody to β-Actin (A2066, Sigma, Stockholm, Sweden), a rabbit monoclonal antibody to phopho-STAT1 (Tyr701) (58D6, Cell Signaling Technology, Danvers, MA) and a rabbit polyclonal antibody to STAT1 (ref. 9172, Cell Signaling Technology, Danvers, Massachusetts, USA).

Techniques: Activity Assay, Injection, Flow Cytometry, Expressing, Modification, Virus

Journal: Bioorganic chemistry

Article Title: Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

doi: 10.1016/j.bioorg.2024.107470

Figure Lengend Snippet: Fig. 3. Impact of indirubins 1 and 2 on the activation status of protein kinase pathways. A-F) Human monocytes were pre-treated with indirubins 1 or 2 at the indicated concentrations, or with inhibitors of GSK-3β (SB216763, 5 µM), CDK8 (CCT-251921, 40 nM), CDK9 (JSH-150, 20 nM) or CDK1 (Ro-3306, 10 µM) for 15 min prior to stimulation with LPS 100 ng/mL for 18 h. (A) Protein expression of β-catenin compared to GAPDH as housekeeping protein. (B) Levels of phosphorylated GSK-3β compared to total expression of GSK-3β protein. (C,D) Levels of phosphorylated STAT1 (C, Ser727 and D, Tyr701) vs. β-actin. (E) Levels of phosphorylated NF-κB (Ser276) compared to GAPDH. (F) Levels of phosphorylated Rb (Ser807/Ser811) vs. β-actin. Protein amounts were evaluated by Western Blot and densito metric analysis thereof. n = 3–4 biological replicates. Statistics: Data are shown as A-E) individual values and means ± SEM. Statistics were calculated by one-way ANOVA for multiple comparisons with Dunnett’s correction, testing treatments against vehicle (0.1 % (v/v) DMSO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit monoclonal anti-COX-2, 1:1000 (12282, Cell Signaling, Danvers, MA), mouse monoclonal anti-β-catenin, 1:1000 (610153, BD Biosciences, San Jose, CA) mouse monoclonal anti-GSK-3β, 1:1000 (9832, Cell Signaling), rabbit monoclonal anti-phospho-GSK-3β (Ser9), 1:1000 (9323S, Cell Signaling), rabbit monoclonal anti-phosphoSTAT1 (Ser727), 1:1000 (ab109461, Abcam, Cambridge, UK), rabbit monoclonal anti-phospho-STAT1 (Tyr701), 1:1000 (ab109457, Abcam), rabbit monoclonal anti-phospho-NF-κB p65 (Ser276), 1:1000 (ab183559, Abcam), rabbit monoclonal anti-phospho-Rb (Ser807/811), 1:1000 (9308S, Cell Signaling), monoclonal anti-β-actin, 1:1000 (4970, Cell Signaling), mouse monoclonal anti-GAPDH, 1:1000 (97166S, Cell Signaling).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 2. Virus infection induces delayed STAT1 Ser-708 phosphorylation. A, WT or IKK/ MEFs were mock-stimulated or stimulated with 100IU/ml IFN-. Protein lysate was collected 16 h post-IFN stimulation and immunoblotted using p-STAT1 Ser-708, p-STAT1 Ser-727, p-STAT1 Tyr-701, and total STAT1 antibodies. B and C, Sendai and WNV virus infections induce STAT1 Ser-708 phosphorylation. HEK293 cells were infected with 100 HA units/ml SenV (B) or West Nile virus strain Madagascar (WNV-MAD) (C) at an m.o.i. of 1. At the indicated times following infection, protein lysates were collected and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, p-IRF-3, total IRF-3, IFIT1, and SenV or WNV. Tubulin and GAPDH were used as loading controls.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Virus, Infection, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE3.TypeI,typeII,andtypeIIIIFNsinduceSTAT1Ser-708phosphor- ylation. 2fTGH cells were mock-treated or treated with 100 IU/ml IFN- (A) or 50 ng/ml IFN- (B). C, PH5CH8 cells were mock-treated (lane 1), treated with 100 ng/ml IFN-1 (lanes 2-6), or 100 IU/ml IFN- (lanes 7-9). Protein lysate was collected at respective time points following IFN treatment and immuno- blotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 4. Signaling through IFNAR is required for STAT1 Ser-708 phosphorylation following type I IFN treatment or virus infection. WT, IRF-3/, or IFNAR/ (A) and parental 2fTGH cells or their derivative U5A cells (which lack IFNAR) (B) were infected with WNV-MAD at an m.o.i. of 1. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, and WNV. C and D, the same cells were also mock-stimulated or stimulated with 100 IU/ml IFN- for 6 or 16 h. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, IFIT2, IFIT3, and IFIT1. Asterisk, nonspecific band.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Phospho-proteomics, Virus, Infection

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 5. STAT1 Ser-708 phosphorylation requires de novo protein syn- thesis, STAT1 tyrosine dephosphorylation, and nuclear export. A, 2fTGH cells were mock-treated (-CHX, lanes 1-5) or treated with CHX (CHX, lanes 6-11) to block protein synthesis. At 30 min (lanes 6-10) or 16 h (lane 11) follow- ing CHX treatment, cells were mock-stimulated (M) or stimulated with IFN-. Cells were harvested at 10 min as well as 1, 6, and 16 h post-IFN stimulation andimmunoblottedtodetectp-STAT1Ser-708,p-STAT1Tyr-701,totalSTAT1, ISG15, and IFIT1. B, 2fTGH cells were not treated (NT, lanes 1-3), pretreated with 100 nM LMB (lanes 4-6), or 50 mM pervanadate (Van, lanes 7-9) 1 h before mock stimulation or stimulation with 100 IU/ml IFN-. Cells were harvested at 1 and 16 h post-stimulation. Immunoblot analysis was performed using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1 antibodies.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Blocking Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 6. IKK mediates IFIT2 expression and protection against WNV pathogenesis in vivo. WT Bl6 and IKK/ mice were mock-infected (PBS only) or infected with 103 pfu of WNV-MAD subcutaneously through footpad injection. A, mice were monitored and scored daily for clinical symptoms over 17 days. Clinical scores from four representative mice per group were graphed. B, spleens from WT or IKK/ mice, mock-infected or infected with WNV-MAD, were collected at days 4, 6, and 12 post-infection. Protein lysates were extracted by homogenizing spleens with radioimmune precipitation assay buffer and immunoblotted using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, IFIT2, IFIT1, WNV, and IKK antibodies. The immunoblot analysis panel is a representative from four mice per infection group.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Expressing, In Vivo, Infection, Injection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 7. A model illustrating that early and late ISGs induction is regulated by multiple STAT1 posttranslational modifications. 1, the canonical JAK-STAT signaling is activated following type I IFN binding to its receptor, which results in STAT1 Tyr-701 phosphorylation, ISGF3 formation, and its nuclear translocation. ISGF3 binding to the ISRE element induces transcription of ISGs. 2, chromatin-bound STAT1 can be phosphorylated by MAPK at residue Ser-727, whichinducesitssumoylation.3and4,nuclearSTAT1isalsoacetylatedbyhistoneacetyltransferase(HAT)CREB-bindingprotein(CBP),resultinginrecruitment of TCP1, which catalyzes STAT1 tyrosine dephosphorylation. Sumoylated-acetylated STAT1 cycles back to the cytoplasm, and both modifications render STAT1 unable to be further tyrosine-phosphorylated. 5 and 6, type I IFN signaling and unknown IFN-stimulated factor(s) activate IKK phosphorylation of STAT1 Ser-708. 7, STAT1 molecules phosphorylated at Ser-708 can enter the nucleus and induce expression of a specific ISG subset. pY, tyrosine phosphorylation; pS, serine phosphorylation; Ac, acetylation; Su, sumoylation.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Binding Assay, Phospho-proteomics, Translocation Assay, Residue, De-Phosphorylation Assay, Expressing

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