Journal: Immunity

Article Title: T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis

doi: 10.1016/j.immuni.2022.12.010

Figure Lengend Snippet: CD226 expression is higher on NK cells in Il22 −/− mice (A) Intravenous model of 4T1-luciferase + (4T1-Luc) metastasis. (B) Representative IVIS images and average radiance from one experiment of two (n = 5). (C) Ex vivo imaging of the lung by IVIS. Linear regression analysis of clonogenic colonies vs. average radiance. (D) Representative dot plots and gating strategy of IFNγ-producing cells in the lungs. Numbers represent the frequency of the parent gate. Frequency of IFNγ + cells in the lung and composition by cell type including CD8 + and CD4 + T cells, NKT cells, and NK cells. (E) Representative dot plots and frequency of IFNγ + NK cells (n = 9 and 10). Data from two independent experiments were pooled. (F) Representative fluorescent microscopy images of lungs from 4T1-injected mice. NKp46 FITC signal is depicted in red, CD8 AF555 in yellow, CD155 PE in green, and Hoechst DNA staining in blue. Intralesional CD155 PE MFI per lesion in WT and Il22 −/− mice. Chip cytometry spatial distribution of NKp46 + (red), CD8 + (green), and CD155 + (gray) cells in the microscopy samples. Frequency of CD8 and NKp46 cells per lesion from the previous graph. Data presented as means ± SEM; p values <0.05 were considered significant by paired t test of one chip of 2 for each condition (n = 3 and 4). (G) Gating strategy and representative plots of CD226 staining on CD8 + T, CD4 + T, NKT, and NK cells. Isotype control is in black. Frequency of CD226 + cells in the lungs (n = 6). Data of one experiment of two. Data presented as means ± SEM; p values <0.05 were considered significant by the Mann-Whitney U test or multiple unpaired t tests with Holm-Sidak correction. See also Figure S6 .

Article Snippet: The following day, FITC conjugated donkey anti-goat antibody (Jackson ImmunoResearch) was applied at a concentration of 10 μg/mL for 2 h at room temperature.

Techniques: Expressing, Luciferase, Ex Vivo, Imaging, Microscopy, Injection, Staining, Chip Cytometry, Control, MANN-WHITNEY

Journal: Immunity

Article Title: T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis

doi: 10.1016/j.immuni.2022.12.010

Figure Lengend Snippet:

Article Snippet: The following day, FITC conjugated donkey anti-goat antibody (Jackson ImmunoResearch) was applied at a concentration of 10 μg/mL for 2 h at room temperature.

Techniques: Purification, Control, Virus, Recombinant, Red Blood Cell Lysis, Transfection, Flow Cytometry, DNA Extraction, Gel Extraction, Plasmid Preparation, cDNA Synthesis, Cell Differentiation, Staining, Cell Isolation, Conjugation Assay, Sequencing, CRISPR, Retroviral, Software

Journal: eLife

Article Title: Epigenetic remodeling by vitamin C potentiates plasma cell differentiation

doi: 10.7554/eLife.73754

Figure Lengend Snippet: ( A ) VC has no effect on IL-21 receptor expression. Mock or VC-treated B cells were cultured for 4 days, and the expression of IL-21R was analyzed by fluorescence-activated cell sorting (FACS) (n = 6). ( B ) VC does not alter the IL-21-induced STAT3 phosphorylation. B cells from day 4 culture were stimulated with IL-21 for 30 min, and the level of STAT3 phosphorylation (pSTAT3) was analyzed by FACS (n = 6). Representative histograms are shown on left and summarized data on right. All data are from at least two independent experiments. Mean ± SEM is shown for bar charts, and the statistical significance was determined by unpaired Student’s t -test. ns, not significant.

Article Snippet: After precleared with ProteinA dynabeads (Thermo Fisher), 20 μg of chromatin was diluted to a final of 500 uL RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation.

Techniques: Expressing, Cell Culture, Fluorescence, FACS, Phospho-proteomics

Journal: eLife

Article Title: Epigenetic remodeling by vitamin C potentiates plasma cell differentiation

doi: 10.7554/eLife.73754

Figure Lengend Snippet: ( A ) Genome browser tracks of the 5hmC enrichment at the Prdm1 locus (mm10 chr10:44,390,000–44,464,000). Data shown are the average of two biological replicates. The locations of E27 and E58 are shown above. ( B ) Differential 5hmC-enriched regions (DhmRs) between the indicated group as in . Colors indicate the differential status of the regions as depicted in the legend. ( C ) DNA methylation in naïve and 48 hr-activated B cells from previous publication ( ; ). The height of the black bars indicates the percentage of CpG methylation, and the red track (CpG) indicates the CpGs that were covered in the analysis. ( D ) VC enhanced STAT3 association at E27. B cells were cultured with or without VC for 4 days as in and treated with or without rmIL-21 (10 ng/mL) for 6 hr. STAT3 binding was analyzed by ChIP-seq. The signals on the genome browser tracks are the average from two biological replicates.

Article Snippet: After precleared with ProteinA dynabeads (Thermo Fisher), 20 μg of chromatin was diluted to a final of 500 uL RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation.

Techniques: DNA Methylation Assay, CpG Methylation Assay, Cell Culture, Binding Assay, ChIP-sequencing

Journal: eLife

Article Title: Epigenetic remodeling by vitamin C potentiates plasma cell differentiation

doi: 10.7554/eLife.73754

Figure Lengend Snippet: ( A ) Genome browser tracks showing the 5hmC enrichment at the Prdm1 locus (same data from ) to show the peak locations. ( B ) Previous published STAT3 ChIP-seq tracks from CD4 T cells stimulated with or without IL-21 are shown . The locations of E27 and E58 are depicted on top. Note that STAT3 binds to E58 in T cells but not in B cells from this study .

Article Snippet: After precleared with ProteinA dynabeads (Thermo Fisher), 20 μg of chromatin was diluted to a final of 500 uL RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation.

Techniques: ChIP-sequencing

Journal: eLife

Article Title: Epigenetic remodeling by vitamin C potentiates plasma cell differentiation

doi: 10.7554/eLife.73754

Figure Lengend Snippet: DNA methylation at E27 was analyzed using bisulfite amplicon sequencing with a Nanopore sequencer. Note that bisulfite sequencing is not able to distinguish 5mC and 5hmC, which usually represents a relatively minor fraction. ( A ) Differential DNA methylation region (DMR) at E27. The DNA methylation (5mC + 5hmC) levels were compared between control (Mock Day4 ) and VC-treated B cells (VC Day4 ). The average DNA methylation ratio at each CpG from three samples is plotted as blue bars (left Y-axis), and the coverage depth from sequencing is plotted as a gray line (right Y-axis). The differential DMR is highlighted with red and CpGs are indicated as red dots. Previously identified STAT3 binding site is highlighted in green. n = 3 for each group. ( B ) VC induces TET-mediated DNA demethylation at E27 DMR. DNA methylation ratios at E27 DMR from indicated groups are shown. Statistical significance was analyzed using a Bayesian hierarchical model with Wald test . WT, n = 3; Tet2/3 -DKO, n = 2. ( C ) TET2 and TET3 are required for the VC-facilitated STAT3 binding to E27. STAT3 binding to E27 was analyzed using ChIP-qPCR. Specific signal from E27 was normalized to the input and then with a background region (‘Materials and methods’). Representative data from one of two experiments are shown (three technical replicates for each group). Statistical significance was calculated using two-way ANOVA, and the relevant comparisons are shown. ***p<0.001, **p<0.01. ns, not significant.

Article Snippet: After precleared with ProteinA dynabeads (Thermo Fisher), 20 μg of chromatin was diluted to a final of 500 uL RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation.

Techniques: DNA Methylation Assay, Amplification, Sequencing, Methylation Sequencing, Control, Binding Assay, ChIP-qPCR

Journal: eLife

Article Title: Epigenetic remodeling by vitamin C potentiates plasma cell differentiation

doi: 10.7554/eLife.73754

Figure Lengend Snippet: ( A ) DNA sequence of mouse Prdm1 E27 DMR. CpGs are labeled in blue. STAT3 binding site and the conserved CpGs between mouse and human are highlighted in gray. ( B ) An element at the 3′ of human PRDM1 with similarity to mouse Prdm1 E27. Note that the sequence shown is from the complementary strand of the reference genome. CpGs are labeled in red. The putative STAT3 binding site and the conserved CpGs are highlighted in gray. ( C ) Sequence alignment between the elements from mouse Prdm1 and human PRDM1. The sequences from ( A ) and ( B ) are aligned with BLAST. Bars indicate identical nucleotides. STAT3 motifs and conserved CpGs are highlighted.

Article Snippet: After precleared with ProteinA dynabeads (Thermo Fisher), 20 μg of chromatin was diluted to a final of 500 uL RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation.

Techniques: Sequencing, Labeling, Binding Assay

Journal: eLife

Article Title: Epigenetic remodeling by vitamin C potentiates plasma cell differentiation

doi: 10.7554/eLife.73754

Figure Lengend Snippet: During plasma cell differentiation, TET2 and/or TET3 is recruited to the enhancers of Prdm1 . In the presence of sufficient VC, TET proteins have a higher enzymatic activity and efficiently oxidize 5mC into 5hmC at certain cis elements, which we termed ‘ascorbate-responsive element’ or E AR . The oxidation and demethylation of 5mC at some E AR may increase the association of transcription factors (TFs), such as STAT3 to E27. However, when VC is limited, the decreased TET activity resulted in an inefficient oxidation of 5mC, which may preclude the binding of TF and/or the maintenance of the association between DNA and nucleosome (not depicted). Therefore, the activity of epigenetic enzyme may reflect the availability of micronutrients or metabolites and may influence gene expression and cell fate decisions.

Article Snippet: After precleared with ProteinA dynabeads (Thermo Fisher), 20 μg of chromatin was diluted to a final of 500 uL RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation.

Techniques: Clinical Proteomics, Cell Differentiation, Activity Assay, Binding Assay, Gene Expression

Journal: eLife

Article Title: Epigenetic remodeling by vitamin C potentiates plasma cell differentiation

doi: 10.7554/eLife.73754

Figure Lengend Snippet:

Article Snippet: After precleared with ProteinA dynabeads (Thermo Fisher), 20 μg of chromatin was diluted to a final of 500 uL RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation.

Techniques: Dot Blot, Enzyme-linked Immunosorbent Assay, Purification, Extraction, Saline, Flow Cytometry, Ligation, Sequencing, Cell Isolation, Isolation, Multiplex Assay, Recombinant, Software

Journal: iScience

Article Title: Caveolin-1 peptide regulates p53-microRNA-34a feedback in fibrotic lung fibroblasts

doi: 10.1016/j.isci.2022.104022

Figure Lengend Snippet:

Article Snippet: Peroxidase-AffiniPure Donkey Anti-Goat IgG , Jackson ImmunoResearch Labs , Cat# 705-035-147; RRID: AB_2313587.

Techniques: Luciferase, Scaffolding, Control, Sequencing, Reverse Transcription, SYBR Green Assay, Staining, Plasmid Preparation, Expressing, Recombinant, shRNA, Software

Journal: eLife

Article Title: Molecular mechanism of Afadin substrate recruitment to the receptor phosphatase PTPRK via its pseudophosphatase domain

doi: 10.7554/eLife.79855

Figure Lengend Snippet:

Article Snippet: Antibody , HRP-conjugated- donkey anti-goat IgG , Jackson ImmunoResearch , Cat#705-035-147 , Western blot: 1:5000.

Techniques: Transfection, Construct, Stable Transfection, Western Blot, Transduction, Recombinant, Mutagenesis, Sequencing, Software, Cell Culture, Lysis, Purification, Magnetic Beads, Chromatography, Protein Concentration, SDS Page, Electrophoresis

Journal: Developmental cell

Article Title: Aging suppresses sphingosine-1-phosphate (S1P) chaperone ApoM in circulation resulting in maladaptive organ repair

doi: 10.1016/j.devcel.2020.05.024

Figure Lengend Snippet: Key Resources Table

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Experimental Models: Organisms/Strains Mouse: Floxed S1pr1 ( Allende et al., 2003 ) RRID: MGI:2681963 Mouse: Apom transgenic ( Christoffersen et al., 2008 ) PMID: 18006500 Mouse: Apom knockout ( Christoffersen et al., 2008 ) PMID: 18006500 Mouse:Cdh5-Cre ERT2 ( Wang et al., 2010b ) RRID: MGI:3848984 Mouse: Albumin-Cre The Jackson Laboratory RRID: IMSR_JAX: 003574 Mouse: S1pr1-Gfp ( Kono et al., 2014 ) PMID: 24667638 Mouse: C57BL6/J The Jackson Laboratory N/A Bacterial and Viral Strains AAV9 ( Gao et al., 2004 ) PMID:15163731 Antibodies Rabbit polyclonal anti-Collagen I Abcam Cat#ab34710 Rabbit polyclonal anti-SMA Abcam Cat#ab5694 Rabbit polyclonal anti-desmin Abcam Cat#ab15200 Goat polyclonal anti-VE-Cadherin R&D Cat#AF1002 Rabbit polyclonal anti-SFTPC Abcam Cat#ab28744 Rabbit polyclonal anti-Aquaporin Abcam Cat#ab78486 Goat polyclonal anti-Podoplanin R&D Cat#AF3244 AF647 donkey anti-rabbit IgG Jackson ImmunoResearch Labs Cat#711-605-152 AF488 donkey anti-rabbit IgG Jackson ImmunoResearch Labs Cat#711-545-152 AF647 donkey anti-goat IgG Jackson ImmunoResearch Labs Cat#705-605-147 AF488 donkey anti-goat IgG Jackson ImmunoResearch Labs Cat#705-545-147 Deposited data RNA Seq raw data This paper GEO Software Image J 1.51 s NIH https://imagej.nih.gov/ij/ Graphpad Prism 8.0 Graphpad Software https://www.graphpad.com/scientificsoftware/prism/ Photoshop Adobe https://www.adobe.com/products/photoshop.html Recombinant DNA ApoM-Fc fusion construct ( Swendeman et al., 2017 ) PMID: 28811382 Chemicals, Peptides, and Recombinant Proteins Tamoxifen Sigma-Aldrich Cat#T5648 Corn Oil Sigma-Aldrich Cat#C8267 Collagenase I Roche Cat#11088793001 Dispase II Roche Cat#04942078001 RIPA Lysis Buffer Santa Cruz Cat#sc-364162 Phosphatase Inhibitor Cocktail Roche Cat#04906837001 Protease Inhibitor Cocktail Roche Cat#04693159001 Lipofectamine™ RNAiMAX Transfection Reagent Invitrogen Cat#13778150 EndoGRO-VEGF Complete Culture Media Kit Millipore Cat#SCME002 DMEM Gibco Cat#11965092 DAPI Invitrogen Cat#D1306 Ketamine Akorn NDC:59399-114-10 Xylazine Akorn NDC:59399-110-20 PFA Electron Microscopy Sciences Cat# 50-980-487 Hydrochloric acid Fisher Scientific Cat# A481-212 Tween80 Sigma-Aldrich Cat#P1754 ApoM-Fc ( Swendeman et al., 2017 ) PMID: 28811382 Critical Commercial Assays Mouse ApoM Elisa Kit Mlbio Biotechnology Cat# {"type":"entrez-nucleotide","attrs":{"text":"ML722506","term_id":"1767879886","term_text":"ML722506"}} ML722506 Hydroxyproline (Hyp) Kit Abcam Cat# ab222941 RNeasy Mini Kit QIAGEN Cat# 74104 EDU Proliferation Kit BD Pharmingen Cat# 565456 Oligonucleotides shRNA targeting sequence: Gankyrin 5’- CCGGG CAGCT TCGAA GAATA GGCAT CTCGA GATGC CTATT CTTCG AAGCT GCTTT TTG -3’ This paper N/A Open in a separate window Key Resources Table

Techniques: Transgenic Assay, Knock-Out, RNA Sequencing Assay, Software, Recombinant, Construct, Lysis, Protease Inhibitor, Transfection, Electron Microscopy, Enzyme-linked Immunosorbent Assay, shRNA, Sequencing

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: NEK2 deficiency decreased the immunosuppressive activity of TAMs and MDSCs (A and B) Flow cytometric analysis showing the percentages of indicated cell populations in the BM and SPL of Nek2 +/+ , Nek2 −/− , 5TGM1/ Nek2 +/+ , and 5TGM1/ Nek2 −/− mice. n = 4–5 mice/group from two independent experiments. Dot plot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (C) Experimental layout. BM-derived macrophages were generated from Nek2 −/− and Nek2 +/+ C57BL/KaLwRij mice after 7 days of M-CSF treatment. TAMs were induced by adding 5TGM1 conditioned medium (CM) for an additional 2 days followed by detection of macrophage survival, cell cycle, CD206 expression, and its effects on T cells proliferation and IFN-γ secretion. (D) Prestoblue staining to detect mouse macrophage survival. n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (E) Cell-cycle analyses of mouse macrophages. n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (F) Flow cytometry analyses of CD206 expression on mouse macrophages. n = 3 independent experiments, each with three technical replicates. (G) Representative histograms and quantification analysis showing the percentages of divided CD8 + T cells co-cultured with TAMs on day 3 (left); IFN-γ levels in the co-culture supernatants on day 1 (right). n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for two-tailed t test. (H) Violin plot showing Mif and Hmgb1 gene expression in MM and plasma cell populations identified in Figure 2 D. (I) Western blots analysis of cell lysates to detect PI3K and STAT3 pathway proteins in mouse TAMs and naive macrophages. (J) Representative histograms and quantification analysis showing the percentages of divided CD8 + T cells co-cultured with MDSCs on day 0 and day 3 (left); IFN-γ levels in the co-culture supernatants on day 1 (right). n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for two-tailed t test. (K and L) Western blots of cell lysates to detect C/EBPβ, arginase-1 (K), as well as PI3K and STAT3 pathway proteins (L) of MDSCs and freshly isolated CD11b + cells. ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Please also see Figure S3 and Table S3 .

Article Snippet: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb , Cell Signaling , Cat# 9145; RRID: AB_2491009.

Techniques: Activity Assay, Comparison, Derivative Assay, Generated, Expressing, Staining, Flow Cytometry, Cell Culture, Co-Culture Assay, Two Tailed Test, Gene Expression, Clinical Proteomics, Western Blot, Isolation

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet:

Article Snippet: Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb , Cell Signaling , Cat# 9145; RRID: AB_2491009.

Techniques: Purification, Recombinant, Western Blot, Stripping, Cell Stimulation, Staining, Cell Isolation, Activation Assay, Extraction, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Reverse Transcription, SYBR Green Assay, RNA Sequencing, Mutagenesis, Knock-Out, Software

Journal: Cancer Prevention Research

Article Title: Garcinol, a Polyisoprenylated Benzophenone Modulates Multiple Proinflammatory Signaling Cascades Leading to the Suppression of Growth and Survival of Head and Neck Carcinoma

doi: 10.1158/1940-6207.capr-13-0070

Figure Lengend Snippet: Figure 3. Garcinol suppresses the phosphorylation and nuclear translocation of STAT3. A, garcinol suppresses p-STAT3 levels in a dose- and time-dependent manner. CAL27 cells (5 105/mL) were treated with the indicated concentrations of garcinol for 120 minutes or treated with 50 mmol/L garcinol for the indicated times, after which whole-cell extracts were prepared and analyzed by phospho-specific and total-STAT3 antibodies. B, garcinol causes inhibition of translocation of STAT3 to thenucleus. CAL27 cells (5 105

Article Snippet: Antibodies to phospho-specific p65 (Ser536), phospho-specific IkBa (Ser32), phospho-specific TGF-b–activated kinase 1 (TAK1; Thr187), TAK1, phosphospecific inhibitor of IkB kinase (IKK)a/b (Ser180/Ser181), IKKa, phospho-specific AKT (Ser473), AKT, phospho-specific mTOR (Ser2448), mTOR, phospho-specific p70S6K (Thr389), p70S6K, phospho-specific STAT3 (Tyr705), STAT3, phospho-specific Src (Tyr416), Src, phospho-specific JAK1 (Tyr1022/1023), JAK1, phospho-specific JAK2 (Tyr1007/1008), JAK2, and CD31 were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Translocation Assay, Inhibition

Journal: Cancer Prevention Research

Article Title: Garcinol, a Polyisoprenylated Benzophenone Modulates Multiple Proinflammatory Signaling Cascades Leading to the Suppression of Growth and Survival of Head and Neck Carcinoma

doi: 10.1158/1940-6207.capr-13-0070

Figure Lengend Snippet: Figure 4. A, garcinol suppresses phospho-Src levels in a time-dependent manner. CAL27 cells (5 105/mL) were treated with 50 mmol/L garcinol, after which whole-cell extracts were prepared and analyzed by Western blotting using phospho- and total-Src antibodies. B, garcinol suppresses phospho-JAK1 levels in a time-dependent manner. The same lysates were analyzed by Western blotting using phospho- and total-JAK1 antibodies. C, garcinol suppresses phospho-JAK2 levels in a time- dependent manner. Whole-cell extracts were analyzed by Western blotting using phospho- and total-JAK2 antibodies. D, garcinol-induced inhibition of STAT3 phosphorylation is reversible. CAL27 cells were treated with 50 mmol/L garcinol for 120 minutes, and washed with PBS to remove the drug before adding fresh medium. Whole-cell extracts were prepared and analyzed by Western blotting using phospho- and total- STAT3 antibodies.

Article Snippet: Antibodies to phospho-specific p65 (Ser536), phospho-specific IkBa (Ser32), phospho-specific TGF-b–activated kinase 1 (TAK1; Thr187), TAK1, phosphospecific inhibitor of IkB kinase (IKK)a/b (Ser180/Ser181), IKKa, phospho-specific AKT (Ser473), AKT, phospho-specific mTOR (Ser2448), mTOR, phospho-specific p70S6K (Thr389), p70S6K, phospho-specific STAT3 (Tyr705), STAT3, phospho-specific Src (Tyr416), Src, phospho-specific JAK1 (Tyr1022/1023), JAK1, phospho-specific JAK2 (Tyr1007/1008), JAK2, and CD31 were purchased from Cell Signaling Technology.

Techniques: Western Blot, Inhibition, Phospho-proteomics

Journal: Cancer Prevention Research

Article Title: Garcinol, a Polyisoprenylated Benzophenone Modulates Multiple Proinflammatory Signaling Cascades Leading to the Suppression of Growth and Survival of Head and Neck Carcinoma

doi: 10.1158/1940-6207.capr-13-0070

Figure Lengend Snippet: Figure 5. Garcinol suppresses STAT3- and NF-kB–regulated gene products involved in proliferation, survival, and angiogenesis and activates caspase-3. The same blots were stripped and reprobed with b-actin antibody to verify equal protein loading (A and B). A, CAL27 cells (5 105/mL) were treated with 25 mmol/L garcinol for indicated time; whole-cell extracts were prepared, separated by SDS–PAGE, and subjected to Western blotting against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, survivin, and VEGF antibodies. B, the same whole-cell extracts were analyzed by Western blotting using caspase-3 and PARP antibodies. C, CAL27 cells (1 106/mL) were treated with 25 mmol/L garcinol for indicated time intervals, after which RNA samples were extracted. One microgram portions of the respective RNA extracts were subjected to reverse transcription to generate corresponding cDNA. Real-time PCR was conducted to measure the relative quantities of mRNA. Each RT product was targeted against cyclin D1, Mcl-1, Bcl-xL, and VEGF TaqMan probes, with 18S as endogenous control for measurement of equal loading of RNA samples. The results were analyzed using Sequence Detection Software version 1.3 provided by Applied Biosystems. Relative gene expression was obtained after normalization with endogenous Hu18S and determination of the difference in Ct between treated and untreated cells using 2DDCt method.

Article Snippet: Antibodies to phospho-specific p65 (Ser536), phospho-specific IkBa (Ser32), phospho-specific TGF-b–activated kinase 1 (TAK1; Thr187), TAK1, phosphospecific inhibitor of IkB kinase (IKK)a/b (Ser180/Ser181), IKKa, phospho-specific AKT (Ser473), AKT, phospho-specific mTOR (Ser2448), mTOR, phospho-specific p70S6K (Thr389), p70S6K, phospho-specific STAT3 (Tyr705), STAT3, phospho-specific Src (Tyr416), Src, phospho-specific JAK1 (Tyr1022/1023), JAK1, phospho-specific JAK2 (Tyr1007/1008), JAK2, and CD31 were purchased from Cell Signaling Technology.

Techniques: SDS Page, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Sequencing, Software, Gene Expression

Journal: Cancer Prevention Research

Article Title: Garcinol, a Polyisoprenylated Benzophenone Modulates Multiple Proinflammatory Signaling Cascades Leading to the Suppression of Growth and Survival of Head and Neck Carcinoma

doi: 10.1158/1940-6207.capr-13-0070

Figure Lengend Snippet: Figure 6. Garcinol inhibits the growth of human HNSCC in vivo. A, athymic mice bearing subcutaneous CAL27 tumors were treated for five times a week for 4 consecutive weeks with 1 and 2 mg/kg garcinol or 0.1% DMSO alone (each group; n ¼ 5). The diameters were measured twice a week for 6 weeks using Vernier caliper, and the tumor volume was calculated using the formula (L x W2)/2, where W is the shortest diameter and L is the longest diameter (n ¼ 6). Data are represented as mean tumor volume SE. , P < 0.05 for control group versus garcinol treatment groups at the end of 4 weeks treatment. B, immunohistochemical analysis of nuclear p65, p-STAT3, Ki-67, and CD31 showed the inhibition in expression of these proteins in garcinol-treated samples. Percentage indicates positive staining for the given biomarker. b.w., body weight.

Article Snippet: Antibodies to phospho-specific p65 (Ser536), phospho-specific IkBa (Ser32), phospho-specific TGF-b–activated kinase 1 (TAK1; Thr187), TAK1, phosphospecific inhibitor of IkB kinase (IKK)a/b (Ser180/Ser181), IKKa, phospho-specific AKT (Ser473), AKT, phospho-specific mTOR (Ser2448), mTOR, phospho-specific p70S6K (Thr389), p70S6K, phospho-specific STAT3 (Tyr705), STAT3, phospho-specific Src (Tyr416), Src, phospho-specific JAK1 (Tyr1022/1023), JAK1, phospho-specific JAK2 (Tyr1007/1008), JAK2, and CD31 were purchased from Cell Signaling Technology.

Techniques: In Vivo, Control, Immunohistochemical staining, Inhibition, Expressing, Staining, Biomarker Discovery

Journal: iScience

Article Title: The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis

doi: 10.1016/j.isci.2022.104386

Figure Lengend Snippet:

Article Snippet: Sections were then washed 10 times with 1X KPBS before incubation in 1X KPBS containing 0.4% Triton and 1:600 dilution of Donkey anti-Goat biotin-SP (Jackson ImmunoResearch) for 1 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Imaging, Recombinant, Modification, Saline, Blocking Assay, Electron Microscopy, Plasmid Preparation, Gel Extraction, Transfection, Polymerase Chain Reaction, Reverse Transcription, Staining, Ligation, Mutagenesis, CRISPR, Cloning, Sequencing, SYBR Green Assay, shRNA, Software

Journal: eLife

Article Title: True S-cones are concentrated in the ventral mouse retina and wired for color detection in the upper visual field

doi: 10.7554/eLife.56840

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Goat Cy3 (Donkey polyclonal) , Jackson Immunoresearch , Cat#705-166-147, RRID: AB_2340413 , IF (1:500).

Techniques: Sequencing, Software