Journal: Stem Cells International

Article Title: Comparison of Stemness and Gene Expression between Gingiva and Dental Follicles in Children

doi: 10.1155/2016/8596520

Figure Lengend Snippet: Specific primer used for quantitative RT-PCR analysis.

Article Snippet: CXCL10 , Positive regulation of leukocyte, chemotaxis , Hs01124251_g1 , 135.

Techniques: Quantitative RT-PCR, Cell Differentiation, Chemotaxis Assay, Activity Assay, Transduction

Journal: Stem Cells International

Article Title: Comparison of Stemness and Gene Expression between Gingiva and Dental Follicles in Children

doi: 10.1155/2016/8596520

Figure Lengend Snippet: Representative genes differentially expressed with higher expression levels in the gingiva than in dental follicles (absolute fold change > 4.0).

Article Snippet: CXCL10 , Positive regulation of leukocyte, chemotaxis , Hs01124251_g1 , 135.

Techniques: Expressing, Standard Deviation, Modification, Activity Assay, Cell Differentiation, Transduction, Chemotaxis Assay

Journal: Stem Cells International

Article Title: Comparison of Stemness and Gene Expression between Gingiva and Dental Follicles in Children

doi: 10.1155/2016/8596520

Figure Lengend Snippet: Verification of microarray results by immunohistochemical (IHC) staining. Hematoxylin-eosin staining in the gingiva (a, f) and dental follicles (DFs) (k, p) (asterisk: outer border neighboring alveolar bone). IHC staining for CXCL10 in the gingiva (b, g) and DFs (l, q). IHC staining for CSTA in the gingiva (c, h) and DFs (m, r). The expression of CXCL10 and CSTA was stained markedly in the gingival epithelium. The IHC staining for AMBN in the gingiva (d, i) and DFs (n, s). AMBN was stained around the outer layer of the DFs. The IHC staining for CXCL12 in the gingiva (e, j) and dental follicles (o, t). CXCL12 was stained in both a cellular layer and the collagenous connective tissue of DFs (scale bars: 200 μ m).

Article Snippet: CXCL10 , Positive regulation of leukocyte, chemotaxis , Hs01124251_g1 , 135.

Techniques: Microarray, Immunohistochemical staining, Immunohistochemistry, Staining, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis.

doi: 10.1016/j.biopha.2021.111881

Figure Lengend Snippet: Fig. 4. Effects of 1 μM As-IV or Oxy on RAC1 activation. RAC1 activation was detected by evaluating the expression of the GTP-RAC1 protein by using a western blot analysis kit. A representative image and the quantification of the band intensity for GTP-RAC1 relative to that of total RAC1 are shown in A-C. (A) Representative western blot analysis of GTP-RAC1 in BMpDCs induced with CCL21 and quantification of the band intensity are shown (5 independent experiments). Data are expressed as the mean ± SE (*p < 0.05, n = 5). Representative western blot analysis of GTP-RAC1 in CCL21-induced migrated BMpDCs following treatment with As- IV (B) or Oxy (C) and the quantification of band intensity are shown (7 independent experiments). Data are expressed as the mean ± SE (*p < 0.05, n = 7). Microarray analysis of BMpDCs induced with a CCL21 gradient following treatment with As-IV or Oxy was performed by using the Clariom S Array Mouse. The differential expression of genes in BMpDCs induced with a CCL21 gradient following treatment with vehicle (control), As-IV or Oxy is displayed in the heatmap (D). The red and blue colors indicated the up-regulation normalized intensity values (log2) and down-regulation normalized intensity values (log2) of each RNA in each sample.

Article Snippet: 30 μm sections cut by using a cryostat (Leica, Nussloch, Germany) were soaked in 0.3% Triton X (Sigma, Missouri, USA) for 2 h and 2% Block Ace (DS Pharma Biomedical, Osaka, Y. Zhang et al. Biomedicine & Pharmacotherapy 141 (2021) 111881 Japan) for 1 h. Then, the colon sections were stained with the primary antibodies rat IgG anti-mouse B220 (1:200, BioLegend, San Diego, CA, USA), hamster IgG anti-mouse CD11c (1:100, BioLegend) and goat IgG anti-mouse CCL21 (1:200, R&D Systems).

Techniques: Activation Assay, Expressing, Western Blot, Microarray, Quantitative Proteomics, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis.

doi: 10.1016/j.biopha.2021.111881

Figure Lengend Snippet: Fig. 6. Effects of As-IV or Oxy on the distribution of CCL21 in the DSS-induced colitis model. The distribution of CCL21 in the colonic ILFs of DSS-induced colitis mice treated with saline (A), As-IV (B) or Oxy (C) was identified by immunohistochemical staining of the colon. Immunohistochemical staining was performed on samples from 3 mice/group, and representative images are presented.

Article Snippet: 30 μm sections cut by using a cryostat (Leica, Nussloch, Germany) were soaked in 0.3% Triton X (Sigma, Missouri, USA) for 2 h and 2% Block Ace (DS Pharma Biomedical, Osaka, Y. Zhang et al. Biomedicine & Pharmacotherapy 141 (2021) 111881 Japan) for 1 h. Then, the colon sections were stained with the primary antibodies rat IgG anti-mouse B220 (1:200, BioLegend, San Diego, CA, USA), hamster IgG anti-mouse CD11c (1:100, BioLegend) and goat IgG anti-mouse CCL21 (1:200, R&D Systems).

Techniques: Saline, Immunohistochemical staining, Staining

Journal: Cancer Research

Article Title: The BH3 Mimetic ABT-737 Induces Cancer Cell Senescence

doi: 10.1158/0008-5472.can-10-1977

Figure Lengend Snippet: Figure 1. Gene transcription changes after ABT-737 treatment. A, microarray analysis of gene expression induced by ABT-737. PV-10 cells were treated in triplicate with DMSO or 10 mmol/L ABT-737 for 24 hours and gene microarray changes documented. B, gene changes associated with senescence. C, qT-PCR analysis of IL-6 and IL-8 transcripts. PV-10 and 22Rv1 cells were treated in triplicate with DMSO, ABT-737 (10 mmol/L) or enantiomer (En., 10 mmol/ L) for 24 hours (mean SD, n ¼ 3). D, the secretion of IL-6 and IL-8 in PV-10 cells treated with DMSO, ABT-737 or enantiomer for 24 hours was determined by ELISA (n ¼ 6, mean SD).

Article Snippet: Conditioned medium was collected following treatment and used at a 2:1 dilution in the human interleukin-6 (IL-6) or IL-8 ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Microarray, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: The proximity-based protein interactome and regulatory logics of the transcription factor p65 NF-κB/RELA

doi: 10.1038/s44319-024-00339-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Both pools were additionally supplemented with assays for three prototypical NF-κB target genes IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1) and CXCL2 (#Hs00236966_m1), two housekeeping genes GUSB (#Hs99999908_m1) and GAPDH (#Hs02758991_g1) and the positive control RELA (#Hs01042019_g1).

Techniques: CRISPR, Bacteria, Recombinant, Cloning, Clone Assay, Mutagenesis, Control, Plasmid Preparation, Sequencing, Luciferase, Gene Expression, Labeling, Binding Assay, Modification, Saline, Western Blot, Transfection, Protease Inhibitor, Random Hexamer, Reverse Transcription, Membrane, In Situ, Proximity Ligation Assay, SYBR Green Assay, Microarray, Software

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: Published ChIP‐seq data from KB cells (Jurida et al , ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig ) or for enhancer or promoter deletions (see Figs and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: ChIP-sequencing, Binding Assay, Sequencing

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: Cross‐linking‐free chromosome conformation capture (i4C) analysis was performed using chromatin from KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Shown are i4C profiles in the 1 Mbp around the CXCL2 locus on chromosome 4 ( ideogram ). Average read counts of two biological replicates are plotted, generated using the CXCL2 promoter ( blue highlight ) or enhancer ( pink highlight ) as a viewpoint. The region of the IL8 promoter/enhancer is also shown ( gray highlight ). Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig software (Williams et al , ) are indicated. All profiles are shown aligned to gene models ( blue ) and CTCF ChIP‐seq, as well as to H3K27ac and H3K4me1 ChIP‐seq data from KB cells (GSE64224 + GSE52470) performed under the same conditions (Jurida et al , ). The breadth of topologically associating domains (TADs) in the locus is indicated above. Meta‐plots showing coverage of ATAC‐seq (this study) and H3K27ac, p65, and RNA polymerase II (RNAPII) ChIP‐seq signals (GSE64224 + GSE52470) at i4C fragments ± 1 kbp contacted by the CXCL2 promoter or enhancer in KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Source data are available online for this figure.

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: Generated, Software, ChIP-sequencing

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: Genome browser views of the CXCL2 and IL8 chemokine loci on human chromosome 4 show H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 and CXCL2 gene models ( blue ). The locations of the deleted NF‐κB binding sites in their flanking enhancer regions are indicated ( orange ). Both loci were mutated using pairs of sgRNAs in stably transfected HeLa cell lines, and Sanger sequencing results of PCR‐amplified genomic regions using DNA of both enhancer‐mutant cell lines (Δp65 eIL8 and Δp65 eCXCL2 ) confirmed removal of 56 and 59 bp, respectively. Blue shades mark the targeted NF‐κB binding sites. mRNA levels of seven IL‐1α‐responsive genes in control (empty vector) or enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines was assessed by RT–qPCR (mean levels ± SEM, normalized to GUSB ; n = 4 (vector, Δp65 eIL8 ), n = 3 (Δp65 eCXCL2 )) at the indicated times after IL‐1α stimulation. *: significantly different to control; P < 0.01, unpaired, two‐tailed Student's t ‐test. Microarray gene expression analysis was performed in HeLa cells ± IL‐1α stimulation for 60 min on control (empty vector; n = 4) and three p65 enhancer‐deletion lines (Δp65 eIL8 , Δp65 e CXCL2 , and Δp65 eIL8+eCXCL2 ; n = 2). Differentially expressed genes were identified based on a moderated t ‐test ( P‐ value < 0.05) and at least threefold change compared to the mean control levels (empty vector). The box plots show distribution of quantile‐normalized mRNA expression values across all experimental conditions and cell lines. Gene sets ( from top to bottom ) represent IL‐1α‐regulated genes, all significantly expressed genes, and all mRNAs expressed from the genes of chromosome 4. Boundaries of the box indicate the 25 th /75 th percentiles, black lines within the box mark the medians, whiskers (error bars) indicate the 10 th /90 th percentiles, and black dots mark the 5 th /95 th percentiles. Additional analyses are provided in Fig B–D. The complete data are provided in Table EV1 . Parental (wt), vector controls, IL8 enhancer‐mutant cells (Δp65 eIL8 ), or stable HeLa lines carrying CRISPR/Cas9‐mediated mutations of the RELA gene (Δ RELA ) and therefore lacking p65 NF‐κB (see also Fig EV4) were left untreated or stimulated with IL‐1α as indicated. Then, total RNA from cell pellets and proteins from supernatants were analyzed by RT–qPCR and ELISA, respectively. IL6 and IL8 mRNA levels are depicted relative to the unstimulated vector controls ( upper panel ). IL‐8 and IL‐6 cytokine levels were normalized to total RNA, and concentrations are shown ( lower panels ). Data are from three independent experiments; shown are means ± SD. Vector controls, IL8 enhancer‐mutant cells (Δp65 eIL8 ), or cells lacking p65 (Δ RELA ) were left untreated or were stimulated with IL‐1α for 8 h in serum‐free cell culture medium. After 7.5 h, half of the cells received puromycin for 30 min to label nascent polypeptides in vivo for monitoring ongoing translation (Iwasaki & Ingolia, ). Then, supernatants were harvested and proteins were precipitated and analyzed for newly synthesized polypeptides by immunoblotting using anti‐puromycin antibodies (left panel ) or for the entire stable secretome by silver staining (right panel ). Shown is one out of two experiments yielding identical results.

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: ChIP-sequencing, Binding Assay, Stable Transfection, Transfection, Sequencing, Amplification, Mutagenesis, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Microarray, Expressing, CRISPR, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vivo, Synthesized, Western Blot, Silver Staining

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: Empty vector controls or Δp65 eIL8 , Δp65 eCXCL2 , and Δp65 eIL8+eCXCL2 enhancer‐mutant HeLa lines were stimulated with IL‐1α for 60 min or were left untreated. Then, actinomycin D (5 μg/ml) was added to stop transcription and RNAs were immediately extracted (reference time point 0 min) or incubations were continued to monitor the decay of IL8 and CXCL2 mRNAs at different time points. RT–qPCR was performed from total RNA, and changes in mRNA expression were calculated and are depicted relative to the 0‐min time point (mean levels ± SEM, normalized to GUSB ; n = 2). Summary of microarray gene expression analysis performed in HeLa cells ± IL‐1α stimulation for 60 min on control (empty vector; n = 4) and three p65 enhancer‐deletion lines (Δp65 eIL8 / ‐ CXCL2 / ‐ IL8+CXCL2 ; n = 2). Differentially expressed genes were identified based on a moderated t ‐test ( P‐ value < 0.05) and at least threefold change compared to the mean control levels (empty vector). All data are provided in Table EV1 . Heatmap depicting fold changes (log 2 ) in response to IL‐1α for each sample over the mean of vector control levels ( blue to red ) for the top 93 IL‐1α‐regulated genes (> 3‐fold, P ‐value < 0.05). Additionally, the effect of the three enhancer‐mutant lines on the IL‐1α response compared to the mean of IL‐1α‐stimulated vector controls is visualized ( purple to green ). Purple shades indicate suppression of IL‐1α‐activated genes. Gene ontology and KEGG terms associated with the 93 shared IL‐1α‐upregulated genes from panel (C) against the entire set of the 14,204 expressed genes.

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Expressing, Microarray

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: NF‐κB (p65), histone marks, and RNA polymerase II enrichment at the IL8 and CXCL2 promoter and enhancer in control (empty vector) or mutated (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines were assessed by ChIP‐qPCR (mean enrichment over input ± SEM) at the indicated times after IL‐1α stimulation. IgG, H3K27ac, H3K4me1, H3, and p65 ChIP‐qPCR data are from three (vector, Δp65 eCXCL2 ) or four (Δp65 EIL8 ) independent experiments performed in duplicate; all others are from at least two independent experiments. *: significantly different to vector controls; P < 0.01, unpaired, two‐tailed Student's t ‐test. As in panel (A), but for the promoters of the IL6 , CCL20 , NFKBIA , CXCL1 , and CXCL3 genes. Data are derived from at least two independent experiments (mean ± SEM, * P < 0.01, unpaired, two‐tailed Student's t ‐test).

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Two Tailed Test, Derivative Assay

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: Representative triple RNA FISH images from HeLa cells ± IL‐1α stimulation for 60 min. Mature mRNAs ( IL8 , β‐actin ; red ) and intronic RNAs ( CXCL2 , purple ; IL8, green ) are detected against nuclei stained with Hoechst 33342 ( blue ). Typical foci marking individually labeled IL8 / CXCL2 transcription sites or merged signals indicating co‐transcription and spatial proximity are enlarged ( inset ). Scale bar: 10 μm. Quantification of RNA FISH signals from parental (wt), control (vector), p65‐deletion (Δp65 eIL8 and Δp65 eCXCL2 ), or p65‐knockout (Δ RELA ) HeLa lines ± IL‐1α stimulation for 60 min. Negative controls (neg ctrl) indicate samples from IL‐1α‐stimulated control cells in which RNA FISH was performed using pre‐amplifier, amplifier, and label probe mixes, but omitting the specific probe sets for IL8 or CXCL2 . These samples were used to define unspecific signals. Data from three independent experiments are pooled and plotted. The box plots show the distributions of FISH signals. Boundaries of the box indicate the 25 th /75 th percentiles, black lines mark medians, and colored lines mark means, respectively. Whiskers (error bars) indicate the 10 th /90 th percentiles, and circles mark all remaining outliers. The data from panel (B) were used to separately quantify the fraction of cells with mono‐ or biallelic IL8 or CXCL2 intronic RNA expression ( purple, green, blue colors ), as well as the extent of colocalizing (overlapping) intronic RNA FISH signals in individual cells, indicating simultaneously activated transcription sites on the same allele ( yellow colors ). The total numbers of cells analyzed are shown above each bar. Data are depicted relative to the total number of analyzed cells. * P < 0.05; Fisher's exact test. Parental (wt) or control HeLa cells (vector) were treated with the TAK1 inhibitor (TAKi) or solvent (DMSO) for 30 min ± IL‐1α stimulation for 60 min. Intronic RNA FISH was performed in three independent experiments and quantified as in panel (C). The total numbers of cells analyzed are shown above each bar. * P < 0.05; Fisher's exact test. As in panel (D), but for human pigmented retinal epithelial cells (RPE‐1) treated with the TAK1 inhibitor (TAKi) or solvent only (DMSO) for 30 min ± IL‐1α stimulation for 60 min. Data information: In panels (D, E), data are pooled from three independent experiments.

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: Staining, Labeling, Plasmid Preparation, Knock-Out, RNA Expression

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: A CRISPR activation (CRISPRa) strategy was applied to test enhancer functions individually. This approach involves a “dead” Cas9 ( blue ) and VP64 ( green ) fusion protein that recruits the NF‐κB ( orange ) and HSF1 ( red ) transactivation domains via MS2 recognition of two stem loops in the sgRNA scaffold ( magenta ). These complexes were targeted to the IL8 or CXCL2 enhancers and promoters (highlights) via different sgRNA pools in HeLa. The position of individual sgRNAs used for CRISPRa is shown in more detail in Fig . Left bar graphs: Wild‐type HeLa cells were transiently transfected with different combinations of plasmids encoding the “dead” Cas9‐VP64 fusion protein, the MS2‐p65‐HSF1 fusion protein, and empty sgRNA vector or versions containing sgRNAs targeting the IL8 enhancer or promoter. Twenty‐four hours post‐transfection, cells were lysed and total RNA was analyzed for expression changes of the indicated genes compared to samples carrying dCas9‐VP64 and MS2‐p65‐HSF1 fusions, but no sgRNAs. Right bar graphs: The same experiments were performed using sgRNAs targeting the CXCL2 enhancer and promoter. Data information: All data are from four independent transfections. Shown are mean values ± SEM. P ‐values are derived from unpaired t ‐tests comparing every condition against cells expressing all transactivators but lacking sgRNAs (first lane in each graph). Only significant differences are marked by asterisks.

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: CRISPR, Activation Assay, Transfection, Plasmid Preparation, Expressing, Derivative Assay

Journal: The EMBO Journal

Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

doi: 10.15252/embj.2019101533

Figure Lengend Snippet: i4C profiles in the 1.6 Mbp around the BMP4 and SAMD4A loci on chromosome 14 ( ideogram ) ± TNFα stimulation for 60 min. Data were generated using the SAMD4A ( blue highlight ) and BMP4 promoters (gray highlight ) or enhancers ( dotted rectangles ) as viewpoints, and profiles are shown aligned to gene models ( blue ) and to ENCODE ChIP‐seq profiles from primary HUVECs. The breadth of topologically associating domains (TADs) in the locus is indicated above ( rectangles ). ΔeBMP4 and ΔeSAMD4A indicate i4C data generated using primers specifically targeting the respective enhancer deletions in the population. Left : electrophoresis profiles of wild‐type (wt) and truncated amplicons ( scissors ) from CRISPR‐edited HUVEC populations, where the upstream BMP4 enhancer was deleted in > 30% alleles. Right : relative changes in nascent (intronic) RNA levels of the BMP4 ( gray ), SAMD4A ( blue ), and CXCL2 TNFα‐inducible genes ( black ) between wild‐type and ΔeBMP4 cell populations in the presence or absence of TNFα stimulation. Shown are mean values ± SD. * P ‐value < 0.05; unpaired, two‐tailed Student's t ‐test ( n = 2). As in panel (B), but for HUVECs carrying ˜12% alleles lacking the SAMD4A intronic enhancer. Shown are mean values ± SD. * P < 0.05; unpaired, two‐tailed Student's t ‐test ( n = 2). Bar plot showing the distribution of split reads that mapped to each chromosome from multi contact (MC)‐i4C experiments using the SAMD4A promoter ( blue ), SAMD4A enhancer ( white ), or BMP4 enhancer ( gray ) as a viewpoint; unmapped reads are also shown. Bar plot showing the number of interacting fragments contained in each analyzed MC‐i4C read. Inset : counts for the number of useful reads and fragments per each viewpoint. MC‐i4C profiles in the 2 Mbp around the BMP4 and SAMD4A loci on chromosome 14 ( ideogram ) ± TNFα stimulation for 60 min. Data were generated using the SAMD4A promoter ( blue highlight ) and enhancer ( dotted rectangle ) or the BMP4 promoter ( gray highlight ) as viewpoints, and profiles are shown aligned to gene models ( blue ) and to ENCODE ChIP‐seq profiles from primary HUVECs. Source data are available online for this figure.

Article Snippet: 2 or 1 μl of this reaction mixture was used to amplify cDNAs using assays on demand (0.25 or 0.5 μl) (Applied Biosystems/Thermo Fisher Scientific) for ACTB (Hs99999903_m1), GUSB (Hs99999908_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1), NFKBIA (Hs00153283_m1), CXCL1 ( Hs00236937_m1), CXCL2 ( Hs00236966_m1), CXCL3 ( Hs00171061_m1), CCL20 ( Hs00171125_m1), and RELA (p65) ( Hs00153294_m1), as well as TaqMan Fast Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific).

Techniques: Generated, ChIP-sequencing, Electrophoresis, CRISPR, Two Tailed Test

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Novel biomarker candidates for OSCC identified using microarray analysis.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Biomarker Discovery, Microarray, Binding Assay

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Expression of CXCL13 mRNA in primary OSCC tissues by RT‐qPCR. (A) CXCL13 expression was higher in tumor tissues compared to adjacent normal tissues in all cases. (B) Significant upregulation of CXCL13 mRNA was observed in tumor tissues. ** p < 0.01 compared to adjacent normal tissues.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum levels of CXCL13 protein in patients with OSCC. (A) The average serum CXCL13 protein in 125 patients with OSCC was 72.8 ρg/mL. (B) The average serum CXCL13 protein in healthy individuals was 33.5 ρg/mL. (C) The reference value determined by the ROC curve for maximizing the TPF/FPF ratio was 53.0 ρg/mL. FPF, false‐positive fraction; TPF, true‐positive fraction.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 levels in patients with OSCC.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Diagnostic sensitivity of serum CXCL13 and SCC in patients with OSCC.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Diagnostic Assay

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 and clinicopathological factors in patients with OSCC. (A) Serum CXCL13 protein levels in T classification. The serum CXCL13 proteins increased in accordance with primary tumor size. The serum CXCL13 levels in T1 were significantly higher than in T3 and T4. (B) The serum CXCL13 proteins in recurrence. The serum levels of CXCL13 proteins in patients with recurrence were significantly elevated. (C) Patients with recurrence were classified into primary recurrence, neck LNM, and distant metastasis (including multiple responses). The serum levels of CXCL13 proteins in neck LNM were significantly elevated. * p < 0.05, ** p < 0.01.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 and prognosis in patients with OSCC. A comparison of overall survival (OS) and disease‐free survival (DFS) in patients with OSCC was made among the two groups, which were classified based on the median serum CXCL13 level using the Kaplan–Meier method with a log‐rank test. High expression of serum CXCL13 indicated poor prognosis in both OS (A, p = 0.044) and DFS (B, p = 0.018) across all stages. In Stage I/II, the groups had no significant difference in OS (C, p = 0.696) and DFS (D, p = 0.435). In Stage III/IV, both OS (E, p = 0.020) and DFS (F, p = 0.009) were significantly poorer in patients with high CXCL13 levels.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Comparison, Expressing

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Visium spatial transcriptome analysis using two cases of primary OSCC tissues. (A) Case 1: Primary tongue SCC tissues were classified into eight clusters based on the gene expression profile. Among these, the tumor area comprised four clusters (Cluster 1, 3, 5, and 6). CXCL13 expression was upregulated in three of four clusters within the tumor area. CD8 expression was detected in all tumor clusters. (B) Case 2: In Case 2, primary tongue SCC tissues were classified into seven clusters. The tumor area consisted of four clusters (Clusters 1, 2, 3, and 6). The expression of CXCL13 and CD8 was upregulated in three of four clusters within the tumor area. CD4 expression was detected only in Cluster 2 in the tumor area but not in Case 1.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Gene Expression, Expressing

Journal: Arthritis Research & Therapy

Article Title: Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study

doi: 10.1186/ar2358

Figure Lengend Snippet: Differentially expressed genes in RASFsn-stimulated chondrocytes (FC ≥ 2; FC ≤ -2; RMA and GCOS)

Article Snippet: Quantitative gene expression was analyzed for chemokine (C-X-C motif) receptor 4 ( CXCR4 , assay ID Hs00607978_s1), thioredoxin interacting protein ( TXNIP , Hs00197750_m1), chondroitin sulfate proteoglycan 2 ( CSPG2 , Hs00171642_m1), IFN-α inducible protein-6–16 ( IFI-6–16 , Hs00242571_m1), cyclooxygenase-2 ( COX-2 , Hs00153133_m1), cartilage oligomeric matrix protein ( COMP , Hs00164359_m1), steroid sulfatase ( STS , Hs00165853_m1) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH , Hs99999905_m1).

Techniques: Binding Assay, Dominant Negative Mutation

Journal: Arthritis Research & Therapy

Article Title: Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study

doi: 10.1186/ar2358

Figure Lengend Snippet: Validation of gene expression of stimulated chondrocytes by real-time RT-PCR. Semi-quantitative real-time RT-PCR of selected genes that were differentially expressed in chondrocytes stimulated with supernatant of a synovial fibroblast cell line derived from a patient with rheumatoid arthritis (RASFsn) as determined by microarray analysis was performed. Real-time RT-PCR gene expression analysis determined that the expression of cyclooxygenase-2 ( COX-2 ), interferon-α inducible protein-6–16 ( IFI-6–16 ) and chemokine (C-X-C motif) receptor 4 ( CXCR4 ) was significantly induced during stimulation of cartilage-like cultures with RASFsn compared with stimulation with supernatant of a synovial fibroblast cell line derived from normal donor (NDSFsn). The gene expression of steroid sulfatase ( STS ), chondroitin sulfate proteoglycan 2 ( CSPG2 ), cartilage oligomeric matrix protein ( COMP ) and thioredoxin interacting protein ( TXNIP ) was significantly repressed during stimulation with RASFsn. Consistent changes were observed between real-time RT-PCR and microarray analysis for all genes examined. The expression of selected genes was calculated as the percentage of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) expression. The mean of each triplicate well of both experimental groups is plotted and the error bars represent SD. For statistical analysis, Students t-test was applied (*, P ≤ 0.05; ***, P ≤ 0.001).

Article Snippet: Quantitative gene expression was analyzed for chemokine (C-X-C motif) receptor 4 ( CXCR4 , assay ID Hs00607978_s1), thioredoxin interacting protein ( TXNIP , Hs00197750_m1), chondroitin sulfate proteoglycan 2 ( CSPG2 , Hs00171642_m1), IFN-α inducible protein-6–16 ( IFI-6–16 , Hs00242571_m1), cyclooxygenase-2 ( COX-2 , Hs00153133_m1), cartilage oligomeric matrix protein ( COMP , Hs00164359_m1), steroid sulfatase ( STS , Hs00165853_m1) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH , Hs99999905_m1).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Derivative Assay, Microarray, Expressing

Journal: Physiological Genomics

Article Title: Gene expression profiling of the effects of organic dust in lung epithelial and THP-1 cells reveals inductive effects on inflammatory and immune response genes

doi: 10.1152/physiolgenomics.00096.2015

Figure Lengend Snippet: TaqMan gene expression IDs for target mRNAs

Article Snippet: TaqMan gene expression IDs for target mRNAs are listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Gene Symbol Gene Name Human Assay ID IL-1β interleukin-1beta Hs01555410_m1 IL-6 interleukin-6 Hs00985639_m1 IL-8 interleukin-8 Hs00174103_m1 ICAM-1 intercellular adhesion molecule-1 Hs00164932_m1 CCL2 chemokine (C-C motif) ligand 2 Hs00234140_m1 CCL5 Hs00982282_m1 Cyr61 Hs00998500_g1 TLR4 toll-like receptor-4 Hs00152939_m1 SOD2 Hs00167309_m1 PTGS2 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) Hs00153133_m1 18S 18S ribosomal RNA Hs99999901_s1 Open in a separate window TaqMan gene expression IDs for target mRNAs DNA microarray analysis.

Techniques: Expressing