Journal: The Journal of Biological Chemistry

Article Title: TET2 has endothelial-specific roles in interferon responses that are dysregulated by hyperglycemia in vitro and in vivo

doi: 10.1016/j.jbc.2025.110520

Figure Lengend Snippet: IFNγ-induced expression of CXCL9, CXCL10 and CXCL11 is decreased by TET2 silencing in HUVEC. A , cytokines with >3-fold decreased ( blue ) or increased ( orange ) abundance in the supernatant of 10 ng/ml IFNγ-treated HUVEC compared to vehicle-treated HUVEC after 24 h. Images show the signal of the five cytokines with the greatest increase or decrease in abundance in vehicle- and IFNγ-treated HUVEC. B-D , concentration of CXCL9 ( B ), CXCL10 ( C ) and CXCL11 ( D ) present in the supernatant of control and TET2 siRNA-treated HUVEC treated with IFNγ for 24 h, measured by ELISA. A Shapiro-Wilk test for normality was performed, followed by an unpaired t test. Data presented as mean ± SEM. n = 3 triplicate samples, representative of three independent experiments. E-H , relative mRNA expression of TET2 ( E ), CXCL9 ( F ), CXCL10 ( G ), and CXCL11 ( H ) in siRNA control-treated and TET2-siRNA-treated HUVEC before and after IFNγ treatment for 24 h, measured by RT-qPCR. A 2-way ANOVA and uncorrected Fisher’s LSD with a single pooled variance was performed. Data presented as mean ± SEM. n = 3 triplicate samples, representative of three independent experiments.

Article Snippet: For CXCL9, CXCL10 and CXCL11, pre-designed Taqman primers/probes (Thermo Fisher) were used (CXCL9 (#Hs00171065_m), CXCL10 (#Hs00171042_m1), CXCL11 (#Hs00171138_m1), β-actin (#Hs99999903_m1)).

Techniques: Expressing, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: TET2 has endothelial-specific roles in interferon responses that are dysregulated by hyperglycemia in vitro and in vivo

doi: 10.1016/j.jbc.2025.110520

Figure Lengend Snippet: 5hmC enrichment at a regulatory element between CXCL9 and CXCL10 in HUVEC following IFNγ treatment. A , CXCL9, CXCL10, and CXCL11 mRNA expression relative to β-actin was determined by qPCR in 10 ng/ml IFNγ-treated HUVEC in the presence or absence of 5-aza-2′deoxycytidine (5azaC), an inhibitor of DNA methyltransferase activity. B , CXCL9 and CXCL10 mRNA expression relative to β-actin was determined by qPCR in HUVEC treated with 10 ng/ml IFNγ for 24 h in the presence or absence of 2-hydroxyglutarate (50 μM each L- and D-2HG). C , the raw signal profiles from hMeDIPseq of vehicle-treated and IFNγ-treated HUVEC were visualized using the UCSC genome browser to assess 5hmC enrichment at the locus spanning CXCL9, CXCL10, and CXCL11 genes. RefSeq genes are displayed. D , inset of region of interest showing peak of 5hmC enrichment and annotations of Weizmann Evolutionary CpG Islands, ENCODE database layered H3K4Me1, H3K4Me3, H3K27Ac, and Dnase I hypersensitivity regions. E , nanopore sequencing read alignments showing CpG hydroxymethylation/methylation motifs marked as open or closed dots, log probability ratios, and smoothed profiles of methylation and hydroxymethylation scores visualized using MethylArtist. Chromosome locations refer to the human genome assembly hg38. Coordinates across the Y-axis refer to methylation bins used in the smoothed methylation profile plot. The yellow shaded region marks the location of 5hmC enrichment observed by hMeDIPseq, and the yellow line denotes its peak, mapped to the nanosequencing profile.

Article Snippet: For CXCL9, CXCL10 and CXCL11, pre-designed Taqman primers/probes (Thermo Fisher) were used (CXCL9 (#Hs00171065_m), CXCL10 (#Hs00171042_m1), CXCL11 (#Hs00171138_m1), β-actin (#Hs99999903_m1)).

Techniques: Expressing, Activity Assay, Nanopore Sequencing, Methylation

Journal: The Journal of Biological Chemistry

Article Title: TET2 has endothelial-specific roles in interferon responses that are dysregulated by hyperglycemia in vitro and in vivo

doi: 10.1016/j.jbc.2025.110520

Figure Lengend Snippet: Overlap in transcriptional dysregulation by TET2 ablation or hyperglycaemia. A-B , IFITM1 ( A ) and ISG15 ( B ) mRNA expression relative to β-actin were determined by qPCR in HUVEC cultured for 1 week in high-glucose (30 mM) or normal-glucose conditions (5 mM) with the addition of 25 mM mannitol as an osmotic control after treatment with 10 ng/ml IFNγ for 24 h followed by its removal for 24 h. C-D , relative mRNA expression of CXCL9 ( C ) and CXCL10 ( D ) in HUVEC cultured for 1 week under high-glucose conditions (30 mM) or normal-glucose conditions (5 mM) with the addition of 25 mM mannitol as an osmotic control, following IFNγ treatment (10 ng/ml) for the final 24 h. E-F , CXCL9 ( E ) and CXCL10 ( F ) mRNA expression relative to β-actin were determined by qPCR in TET2-silenced HUVEC treated with 10 ng/ml IFNγ for 24 h in the presence or absence of high glucose (30 mM) or normal glucose conditions (5 mM) with the addition of 25 mM mannitol as an osmotic control. G , overlap between downregulated genes in CD31+ cells from TET2 KO mice (compared to WT mice) or diabetic mice induced by HFD consumption (compared to chow-fed mice). H , overlap between upregulated genes in CD31+ cells from TET2 KO mice (compared to WT mice) or diabetic mice induced by HFD consumption (compared to chow-fed mice). Differentially expressed genes (DEGs) were defined as genes with p < 0.05. I) Pathway analysis was performed on overlapping DEGs from ( C ) and ( D ). The top 10 pathways predicted to be activated are displayed in order of −log ( p -value) calculated by Fisher’s Exact Test. Darker orange bars represent higher z-scores.

Article Snippet: For CXCL9, CXCL10 and CXCL11, pre-designed Taqman primers/probes (Thermo Fisher) were used (CXCL9 (#Hs00171065_m), CXCL10 (#Hs00171042_m1), CXCL11 (#Hs00171138_m1), β-actin (#Hs99999903_m1)).

Techniques: Expressing, Cell Culture, Control

Journal: Communications Biology

Article Title: Reshaping tumor microenvironment by regulating local cytokines expression with a portable smart blue-light controlled device

doi: 10.1038/s42003-024-06566-y

Figure Lengend Snippet: a Schematics of light-controlled gene expression system and plasmids constructions. b Regulation of gene expression with light intensities from 0 to 1000 μW cm -2 with a customized 24-well plate light-controllable hardware, and c the intensity-response curve of mRuby expression in P815-M-ILs cells at 48 h (flow cytometry assay). d Time-dependent mRNA expression levels of Ifng and Cxcl10 (qPCR assay, normalized to time zero) in P815-IFNG cells and P815-M cells (control group). e Time-dependent production of murine IFNG and CXCL10 proteins (ELISA assay) under the continuous illuminating (On), dark control (Off) and alternative illuminating group (12/12 h light/dark cycles, 12 h O/N). Flow cytometry data is a representative of three independent experiments, each with similar results. Other data are shown as mean ± sem ( n = 3). All statistical significant were computed against the control group, with * P < 0.05; ** P < 0.01.

Article Snippet: Cell supernatants from in vitro studies were collected at designed time points (0, 12, 24, 48, and 72 h after illumination) to quantify cytokines and chemokines with murine CXCL10 and IFNG ELISA kit (BOSTER, China).

Techniques: Gene Expression, Expressing, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Reshaping tumor microenvironment by regulating local cytokines expression with a portable smart blue-light controlled device

doi: 10.1038/s42003-024-06566-y

Figure Lengend Snippet: a Timeline of in vivo experiment using DBA/2 mice with matched bilateral tumors (Left: P815-IFNG; Right: P815-M). b During illumination, representative BLI images of bilateral tumor-bearing mice at different times (Day 0, Day 3, and Day 6). c – d Representative BLI images of bilateral tumor-bearing mice were taken on different days (Day 0, Day 3, and Day 6) after illumination. Tumor size was quantified with average radiance value and compared with BLI signal at day 0 (the first day of illumination). e – g Blood analyses were performed after 6-day illumination, and the results show changes in the number of white blood cells (WBC) e , lymphocytes f , neutrophils g , monocytes ( h ) between light (2.5 mW cm -2 ), dark and control groups. i – l The concentration of murine INFG i , CXCL10 j , IL-6 k , and IL-10 ( l ) in plasma by the end of day 6 under illumination. Data are shown as mean ± s.e.m ( n = 4). Statistic significant was computed against the control group as * P < 0.05; ** P < 0.01.

Article Snippet: Cell supernatants from in vitro studies were collected at designed time points (0, 12, 24, 48, and 72 h after illumination) to quantify cytokines and chemokines with murine CXCL10 and IFNG ELISA kit (BOSTER, China).

Techniques: In Vivo, Control, Concentration Assay, Clinical Proteomics

Journal:

Article Title: Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-?B

doi:

Figure Lengend Snippet: IP-10 expression in REC cells following infection with adenovirus vectors. (A) REC cells infected with 5 × 1010 OPU of adCMVβgal per ml express IP-10 protein at 16 h as detected by ELISA (6.1 ± 1.1 versus 50.3 ± 5.7 ng/ml; P < 0.0002). (B) IP-10 mRNA expression detected by RNase protection assay in REC cells following stimulation with IFN-γ. At 6 h, adCMVβgal induces the expression of IP-10 more than stimulation with 1 U/ml but less than stimulation with 10 U of IFN-γ per ml. Bars represent the SD. (C) AdCMVβgal, adCMV-GFP, FG140, and UV-psoralen-inactivated adenovirus (UV/Ps-Ad) induce the expression of IP-10 mRNA in REC cells 6 and 16 h following infection as seen by RNase protection. Vehicle-treated cells do not induce the expression of IP-10.

Article Snippet: The protein concentration was measured against a recombinant mouse IP-10 standard (R&D Systems).

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Rnase Protection Assay

Journal:

Article Title: Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-?B

doi:

Figure Lengend Snippet: Adenovirus vector-induced activation of the IP-10 promoter in REC cells. (A) Organization of the 237-bp fragment upstream of the transcription start site of the mouse IP-10 gene. Positions of the deletional mutants are noted in relation to the potential cis elements involved. (B) Luciferase reporter assay. AdCMVβgal (■) activates the 533-bp fragment of the IP-10 promoter >10-fold more than the vehicle (□)-treated cells. AdCMVβgal-induced luciferase activity is diminished but still significantly greater than with the vehicle in REC cells transfected with IP-10 promoter deletion constructs pGL3–IP-10(−237), pGL3–IP-10(−190), and pGL3–IP-10(−161). Luciferase activity falls to baseline levels in REC cells transfected with the deletional mutant pGL3–IP-10(−96), thus confirming that the minimal elements responsible for IP-10 promoter activation are located between positions −161 and −96 of the IP-10 5′-flanking region. Bars represent the SD.

Article Snippet: The protein concentration was measured against a recombinant mouse IP-10 standard (R&D Systems).

Techniques: Plasmid Preparation, Activation Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Construct, Mutagenesis

Journal:

Article Title: Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-?B

doi:

Figure Lengend Snippet: EMSA of REC cell nuclear extracts. (A) Nuclear extracts from untreated and vehicle-treated REC cells constitutively form DNA-protein complexes C1 and C2 when incubated with the radiolabeled oligonucleotide, IP-10(−124/−94). IP-10(−124/−94) corresponds to base pairs −124 to −94 of the IP-10 5′-flanking region and contains the NF-κB sequence GGGACTTCC at position −113. AdCMVβgal significantly increases C1 complex 6 and 12 h following infection in REC cells. (B) EMSA using radiolabeled DNA fragment IP-10(−124/−94) and REC nuclear extracts following infection with 5 × 1010 OPU of various adenovirus vectors per ml. C1 complex is increased by adCMV-GFP, FG140, and UV-psoralen-inactivated adenovirus (UV/Ps-Ad) 6 h following infection, a result similar to the pattern induced by adCMVβgal. C2 complex is minimally or not increased significantly over baseline levels following infection with different adenovirus vectors.

Article Snippet: The protein concentration was measured against a recombinant mouse IP-10 standard (R&D Systems).

Techniques: Incubation, Sequencing, Infection

Journal:

Article Title: Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-?B

doi:

Figure Lengend Snippet: Competitor EMSA using nuclear extracts from adCMVβgal-infected REC cells and radiolabeled probe IP-10(−124/−94). C1 complex is not competed away by the nonspecific DNA fragment (Oligo 1) or by a DNA fragment containing an alternate NF-κB sequence GGGATGCCC (Oligo 2). DNA-protein interaction is competed for effectively with unlabeled IP-10(−124/−94), confirming the specificity of this interaction.

Article Snippet: The protein concentration was measured against a recombinant mouse IP-10 standard (R&D Systems).

Techniques: Infection, Sequencing

Journal:

Article Title: Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-?B

doi:

Figure Lengend Snippet: Adenovirus vector-induced activation of NF-κB. (A) EMSA using nuclear extracts from adCMVβgal-infected REC cells and radiolabeled probe IP-10(−124/−94). Anti-NF-κB p65 supershifts C1 complex, whereas anti-NF-κB p50 supershifts C2 complex, confirming the activation of p65 primarily by adenovirus vectors. NF-κB p50 is present in the nuclear extracts but is minimally increased by adenovirus vectors compared to untreated or vehicle-treated cells. (B) RNase protection assay of REC cell RNA following infection with 5 × 1010 OPU of adCMVβgal or adCMV-IκBα per ml. Unlike other adenovirus vectors, adCMV-IκBα does not significantly induce the expression of IP-10 mRNA at 6 and 16 h following infection. Coinfection of adCMVβgal and adCMV-IκBα results in suppression of adCMVβgal-induced expression of IP-10 mRNA at 6 and 16 h, confirming the importance of NF-κB in mediating the transcription of IP-10 after infection with adenovirus vectors.

Article Snippet: The protein concentration was measured against a recombinant mouse IP-10 standard (R&D Systems).

Techniques: Plasmid Preparation, Activation Assay, Infection, Rnase Protection Assay, Expressing

Journal:

Article Title: Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-?B

doi:

Figure Lengend Snippet: RNase protection assay of REC cells infected with adCMVβgal. IP-10 mRNA is induced by adCMVβgal as early as 3 h following infection in the absence of TNF-α and IFN-γ. RNA from DBA/2 mouse liver harvested 16 h following infection with 2 × 1011 OPU of adCMVβgal is used as a positive control for TNF-α and IFN-γ expression.

Article Snippet: The protein concentration was measured against a recombinant mouse IP-10 standard (R&D Systems).

Techniques: Rnase Protection Assay, Infection, Positive Control, Expressing