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: 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: Gene

Article Title: Role for IL-10 in the transcriptional regulation of Roquin-1

doi: 10.1016/j.gene.2014.07.056

Figure Lengend Snippet: Transcriptional regulation of Rc3h1. (A) A web-based search for transcriptional factors that bind to the Rc3h1 promoter identified binding sites for STATs, GATA, IKZF and c-Rel. Luciferase reporter assays were used to evaluate the activational effects of eight transcription factors on the Rc3h1 promoter regions. Using a (B) -2.1 kb Rc3h1 promoter region, STAT1, STAT3, GATA2, and c-Rel resulted in a significant increase in luciferase activity; IKZF2 resulted in a significant decrease. STAT1, STAT3, GATA2, and c-Rel also resulted in increased luciferase activity using the (C) -1.3 kb and (D) -0.2 kb Rc3h1 promoter regions. Values are fold induction of luciferase activity of transcription factor-transfected cells relative to non-transcription factor transfected cells. Data are mean values ± SEM of 3-4 independent experiments.

Article Snippet: The next day, cells were transfected using Fugene HD (Promega, Madison, WI) with 200 ng of a Rc3h1 promoter luciferase reporter plasmid cloned into the pGLuc-Basic plasmid (New England Biolabs, Ipswich, MA) by our laboratory, along with 100 ng of a plasmid encoding for either constitutively-active STAT1 (eGFP STAT1 S727E, Addgene, Cambridge, MA), constitutively-active STAT3 (Stat3-C Flag pRc/CMV, Addgene), constitutively-active STAT5 (CA-STAT5-GFP-RV ( Yang et al., 2011 ), a gift of Dr. Jinfan Zhu), c-Rel (c-Rel cFlag pcDNA3, Addgene), GATA2 (pFlag-GATA2, Addgene), IKZF1, IKZF2, and IKZF3 transcription factors (cloned into the pEF6/V5-His plasmid in our laboratory), and 100 ng of a β-galactosidase plasmid (pSV-β-galactosidase).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection

Journal: Gene

Article Title: Role for IL-10 in the transcriptional regulation of Roquin-1

doi: 10.1016/j.gene.2014.07.056

Figure Lengend Snippet: ChIP assays showing GATA2 interactions with: (A) -2.2 kb, (B) -1.8 kb, (C) -1.0 kb, and (D) -0.4 kb RC3H1 promoter regions. Data in each panel represent one ChIP experiment; the average values of all four data entries for the GATA2 ChIP assays (anti-GATA2 antibody) was significantly greater (p<0.001) than that of ChIP assay results done with None (mock) transfections, or STAT1 transcription factor transfections. (E, F) ChIP assays showing the effect of IL-10 on transcription factor interactions with the Rc3h1 promoter.

Article Snippet: The next day, cells were transfected using Fugene HD (Promega, Madison, WI) with 200 ng of a Rc3h1 promoter luciferase reporter plasmid cloned into the pGLuc-Basic plasmid (New England Biolabs, Ipswich, MA) by our laboratory, along with 100 ng of a plasmid encoding for either constitutively-active STAT1 (eGFP STAT1 S727E, Addgene, Cambridge, MA), constitutively-active STAT3 (Stat3-C Flag pRc/CMV, Addgene), constitutively-active STAT5 (CA-STAT5-GFP-RV ( Yang et al., 2011 ), a gift of Dr. Jinfan Zhu), c-Rel (c-Rel cFlag pcDNA3, Addgene), GATA2 (pFlag-GATA2, Addgene), IKZF1, IKZF2, and IKZF3 transcription factors (cloned into the pEF6/V5-His plasmid in our laboratory), and 100 ng of a β-galactosidase plasmid (pSV-β-galactosidase).

Techniques: Transfection

Journal: Gene

Article Title: Role for IL-10 in the transcriptional regulation of Roquin-1

doi: 10.1016/j.gene.2014.07.056

Figure Lengend Snippet: IL-10 increased Rc3h1 transcription factor expression. (A) EL4 cells were cultured in the presence of mouse rIL-10, or rTGFβ as a control cytokine. Gene expression was measured at 0, 24, and 48 hr. Compared to the rTGFβ control cytokine, exposure of cells to IL-10 resulted in a significant increase in gene expression of STAT1, STAT3, GATA2, and c-Rel, as well as a non-statistical increase in IKZF2 48 hr post-IL-10 treatment. Data are mean values ± SEM of 3 experiments. (B) EL4 cells were transfected with siRNAs to either STAT1, STAT3, GATA2, c-Rel, IKZF2, a scramble control siRNA, or no siRNA for 4 hr followed by exposure to rIL-10 as described in the Materials and Methods. Cells were harvested and Rc3h1 expression was measured by qRT-PCR. All transcription factor-specific siRNAs decreased Rc3h1 expression relative to the control siRNA-transfected cells. For STAT1 and STAT3, this occurred in a statistically-significant manner. (C) Efficiency of knockdown of transcription factor expression. Data are mean values ± SEM of 3 experiments. ** p<0.01, * p<0.05, ◆ p<0.1 and >0.05. (D) A model whereby IL-10 exerts an effect on the transcriptional regulation of Rc3h1.

Article Snippet: The next day, cells were transfected using Fugene HD (Promega, Madison, WI) with 200 ng of a Rc3h1 promoter luciferase reporter plasmid cloned into the pGLuc-Basic plasmid (New England Biolabs, Ipswich, MA) by our laboratory, along with 100 ng of a plasmid encoding for either constitutively-active STAT1 (eGFP STAT1 S727E, Addgene, Cambridge, MA), constitutively-active STAT3 (Stat3-C Flag pRc/CMV, Addgene), constitutively-active STAT5 (CA-STAT5-GFP-RV ( Yang et al., 2011 ), a gift of Dr. Jinfan Zhu), c-Rel (c-Rel cFlag pcDNA3, Addgene), GATA2 (pFlag-GATA2, Addgene), IKZF1, IKZF2, and IKZF3 transcription factors (cloned into the pEF6/V5-His plasmid in our laboratory), and 100 ng of a β-galactosidase plasmid (pSV-β-galactosidase).

Techniques: Expressing, Cell Culture, Control, Gene Expression, Transfection, Quantitative RT-PCR, Knockdown