Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A and B: bulk RNA-seq of aortic tissues from control and AD mice (n=3 per group). Volcano plot displaying differentially expressed genes in aortas between the two groups. Cthrc1 was significantly up-regulated in AD mouse models. (B) KEGG enrichment analysis of differentially expressed genes in aortic tissues from control and AD mice. (C) C to F: scRNA-seq data of 4 samples from control and AD mice. Violin plots showing nFeature, nCount, percent.mt and expression level of the housekeeping gene beta-actin (Actb) after quality control of 4 samples. (D) UMAP plot of 82,326 single cells from 4 samples colored according to the 15 clusters. (E) Heatmap showing expression signatures of the marker genes in each cell type. (F) Heatmap showing expression signatures of the top differentially expressed genes in 2 fibroblast subclusters. (G) UMAP plots showing CTHRC1 expression in fibroblasts from control individuals and patients with ascending thoracic aortic aneurysm (GSE155468). (H) H to J: CTHRC1 protein levels in vitro (NIH3T3 cells (H), human umbilical vein endothelial cells (HUVECs) (I), and MOVAS cells(J), incubated with Ang-II (10^-6 M) and BAPN (0.2 mM). (K) bulk RNA sequencing of aortas in male and female mice. PCA of gene profiles of transcriptome between different sexes. (L) Volcano plot displaying differentially expressed genes in aortic tissues from control and AD female mice (n=3 per group). Cthrc1 was also significantly up-regulated in AD female mouse models. (M) KEGG enrichment analysis of the differentially expressed genes in AD female mouse models. (N) Immunohistochemical staining for CTHRC1 in female mouse aortas. The orange arrows indicate CTHRC1 expression in adventitial fibroblasts of the dissected aortas. (O) Female mouse serum CTHRC1 content in control and AD mice (n=6). (an unpaired two-tailed Student’s t-test, **** P ≤ 0.0001.)

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: RNA Sequencing, Control, Expressing, Marker, In Vitro, Incubation, Immunohistochemical staining, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A schematic diagram illustrating the experiment design. Single-cell RNA sequencing was applied to 4 samples from C57BL/6 mice that were infused with saline or simultaneously administrated by Ang-II/BAPN for 14 days. (B) UMAP plot of the 82,326 single cells from the 4 samples outlined in (A), showing the identification of 9 main clusters. (C) The proportion of 9 cell clusters in control and AD mice. (D) UMAP plot of the 9,043 fibroblasts with 2 subclusters. (E) UMAP plots showing comparison of fibroblast clusters or Cthrc1 expression between the AD and control group. (F) Immunohistochemical staining for CTHRC1 in mouse aortas. The orange arrows indicating CTHRC1 expression in adventitial fibroblasts of the dissected aortas. (G) Representative images showing immunoblotting analysis of CTHRC1 levels in control and AD mice (n=6). (H) Mouse serum CTHRC1 content in control and AD groups (n=6). Data are presented as mean ± SEM. (Student’s t test, ** P ≤ 0.01.) (I) Heatmap of expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly between control and AD mice. (J) Scatterplot showing gene expression level in the adventitia between A+ Pos and A+ Neg samples. Red: upregulated genes in the adventitia of aortas from patients prone to aorta rupture, blue: downregulated genes. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: RNA Sequencing, Saline, Control, Comparison, Expressing, Immunohistochemical staining, Staining, Western Blot, Gene Expression

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Experiment design. 6-week-old male Cthrc1 -/- mice (n=16) and wide type (WT) mice (n=18) were treated with saline or Ang-II/BAPN for 14 days. (B) Representative images of immunoblotting analysis of CTHRC1 levels in aortas from the mice in A. Bottom: quantification of CTHRC1 expression in aortas. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01.) (C) Representative ultrasound images of aorta in mice (scale bar, 1mm). (D) Measurements of maximum aortic diameter of mice in A (n=6-9 per group). (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01, *** P ≤ 0.0001.) (E) Representative pictures of whole aortas from the mice in A. The yellow arrows indicate the dissected sites of vascular lesions. (F) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas from WT and Cthrc1 -/- mice. (Fisher’s exact test, * P ≤ 0.05.) (G) Kaplan-Meier survival rates analysis of the mice in A. (Kaplan-Meier survival rates analysis, * P ≤ 0.05, ns, no significance.) (H) Representative images of histological staining with hematoxylin and eosin in aortic sections from the mice in A. Right: measurements of aortic wall thickness of aortas (scale bar, 50 μm). (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01.) (I) Representative images of Elastica van Gieson staining in aortic sections from the mice in A. Right: measurement of numbers of elastin breaks per vessel (scale bar, 100 μm). (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, *** P ≤ 0.0001). (J) UMAP plot of the 28,700 single cells from WT and Cthrc1 -/- mice challenged with Ang-II/BAPN for 14 days, displaying the identification of 8 major clusters. (K) The proportion of 8 cell clusters in WT and Cthrc1 -/- mice infused with Ang-II/BAPN. (L) Upper: UMAP plot of fibroblast subclusters in the WT group and Cthrc1 -/- group respectively. Bottom: the proportion of FB1 and FB2. (M) KEGG enrichment analysis of differentially expressed genes in aortic tissues from WT and Cthrc1 -/- mice analyzed by bulk-RNA seq data. (N) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Saline, Western Blot, Expressing, Comparison, Staining, RNA Sequencing

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Immunohistochemical staining for CTHRC1 in aortas from the Cthrc1 -/- and WT mice. The orange arrows indicate CTHRC1 expression in adventitial fibroblasts of the dissected aortas, with no positive staining observed in Cthrc1 -/- group. (B) Alican blue staining of aortas from the Cthrc1 -/- and WT mice. (C) C to F: bulk RNA-seq of aortic tissues from WT and Cthrc1 -/- mice. GSEA showing the inhibition of extracellular matrix disassembly and the activation of vascular smooth muscle contraction. (D) : D to F: Violin plots illustrating gene expressions of MMPs (D), ADAMs (E) and ADAMTSs (F) between WT and Cthrc1 -/- mice infused with Ang-II/BAPN. MMPs: matrix metalloproteinases; ADAMs: a disintegrin and metalloproteinases; ADAMTSs: ADAMs with a thrombospondin motif. (G) G to P: 6-week-old female Cthrc1 -/- mice (n=16) and WT mice (n=18) were treated with saline or Ang-II/BAPN for 14 days. Representative ultrasound images of aorta in female mice (scale bar, 1mm). (H) Measurements of maximum aortic diameter of mice in G (n=8 per group). (a Welch ANOVA test followed by a post hoc analysis using the Tamhane T2 method, *** P ≤ 0.001; ** P ≤ 0.01.) (I) Representative pictures of whole aortas from the female mice. The yellow arrows indicate the dissected sites of vascular lesions. (J) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas from WT and Cthrc1 -/- female mice. (Fisher’s exact test, * P ≤ 0.05.) (K) Kaplan-Meier survival rates analysis of the four groups of female mice. (Kaplan-Meier survival rates analysis, ns, no significance.) (L) Immunohistochemical staining for CTHRC1 in aortas from the female mice. The orange arrows indicate CTHRC1 expression in adventitial fibroblasts of the dissected aortas from the WT female mice, with no positive staining observed in Cthrc1 -/- female mice (scale bar, 50 μm). (M) Representative images of histological staining with hematoxylin and eosin in aortic sections from the female mice (scale bar, 50 μm). (N) Measurements of aortic wall thickness of aortas. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, *** P ≤ 0.001; **** P ≤ 0.0001.) (O) Representative images of EVG staining in aortic sections from the female mice (scale bar, 50 μm). (P) Measurements of numbers of elastin breaks per vessel. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01; *** P ≤ 0.001.) (Q) Q to S: bulk RNA-seq of aortic tissues from WT and Cthrc1 -/- female mice. PCA of gene profiles of transcriptome between different sexes. (R) KEGG enrichment analysis of differentially expressed genes in aortic tissues from WT and Cthrc1 -/- female mice. (S) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Inhibition, Activation Assay, Saline, Comparison

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Comparison of the total number of cell-cell interactions and the interaction strength from the scRNA-seq data of control and AD mice. (B) Circle plot showing differential cell-cell interaction numbers and strength between fibroblast, SMC, EC, MoMaphDC, Schwann, B cell and T cell clusters predicted by CellChat. Each circle represents one cell cluster, edges between circles represent intercellular signaling between cell clusters, and edge thickness reflects interaction number or strength. Red edges represent increased interaction number or strength and blue represent decreased interaction number or strength in AD mice versus control mice. (C) Identification of up-regulated signaling by comparing the communication probabilities mediated by ligand–receptor pairs from fibroblasts to SMCs in AD mouse models. (D) D to H: scRNA-seq of the two aortas from WT and Cthrc1 -/- mice infused with Ang-II/BAPN for 14 days were pooled per group. The proportion of major cell types including SMCs, ECs and FBs in Ang-II/BAPN-infused WT and Cthrc1 -/- mice. (E) Bar diagram showing the percentage of 4 subclusters in aortic SMCs from the two groups of mice. (F) Boxplot illustrating the differential gene expression of contractile genes in aortic SMCs of the two mouse groups. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.0001. ns, no significance.) (G) Barplot depicting the numbers of differential expressed genes in SMC subclusters of the two mouse groups. (H) Gene Ontology enrichment analysis of down-regulated genes in fibromyocytes of the two mouse groups. (I) Immunofluorescence staining of α-SMA and SM22α expression in aortas from Ang-II/BAPN-infused WT and Cthrc1 -/- mice. DAPI: 4’,6-diamidino-2-phenylindole (scale bar: 100 μm). (J) Quantification of α-SMA and SM22α positive area in aortas. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, **** P ≤ 0.00001.) (K) K to L: Incubating the murine aortic smooth muscle cells (MOVAS) with rhCTHRC1 in vitro. Gene Ontology enrichment analysis of up-regulated genes in MOVAS cells treated with rhCTHRC1 by bulk RNA-seq. (L) Heatmap showing the expression levels of genes related to metallopeptidase activity, activation of MAPKK activity and positive regulation of smooth muscle cell proliferation in MOVAS cells treated with rhCTHRC1 or Vehicle. (M) Immunoblotting analysis of the protein levels of α-SMA, SM22α, and p-ERK1/2 in MOVAS with or without rhCTHRC1 treatment. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Comparison, Control, Gene Expression, Immunofluorescence, Staining, Expressing, In Vitro, RNA Sequencing, Activity Assay, Activation Assay, Western Blot

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A to G: scRNA-seq data of aortas from WT and Cthrc1 -/- mice challenged with Ang-II/BAPN for 14 days. Violin plots showing data characteristics on nFeature, nCount and percent.mt after quality control of the two groups. (B) The violin plot showing the expression level of the housekeeping gene beta-actin (Actb) between the two groups. The relative stability of Actb expression indicates consistency. (C) UMAP plot of 82,326 single cells from the samples colored according to the 14 clusters. (D) Heatmap showing expression signatures of the marker genes in each cell type. (E) UMAP plot of 1,962 SMCs colored according to the identified 4 SMC subclusters. (F) Heatmap showing the scaled mean expression of signature genes in 4 SMC subclusters. (G) Gene Ontology enrichment analysis of up-regulated genes in contractile SMCs of the two groups of mice. (H) Immunoblotting analysis of α-SMA and SM22α expression in aortas from Ang-II/BAPN-infused WT and Cthrc1 -/- mice (n=6). (I) Quantification of α-SMA and SM22α protein expression levels. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, *** P ≤ 0.001, **** P ≤ 0.0001.) (J) J and K: whole transcriptome analysis of MOVAS cells treated with vehicle and rhCTHRC1. A volcano plot displaying differentially expressed genes. Upward expression of Dcn , Adam9 , Angpt2 and Mmp8 gene in the presence of rhCTHRC1. (K) KEGG enrichment analysis of up-regulated genes in aortas from Ang-II/BAPN-infused WT and Cthrc1 -/- mice.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Control, Expressing, Marker, Western Blot, Comparison

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Workflow of the immunoprecipitation (IP) of CTHRC1 identified by mass spectrometry (MS). (B) Venn diagram of intersection analyses of two independent assays for potential receptors of CTHRC1 on MOVAS cells. Right: Scores of candidate binding proteins identified by MS. (C) Co-IP-blotting of CTHRC1 with ADAM9 receptor on MOVAS cells. (D) D to E: whole transcriptome analysis of MOVAS cells incubated with rhCTHRC1, knockouting Roas26 (control) or Adam9 ( Adam9 -KO). Gene Ontology enrichment analysis of down-regulated genes in Adam9 -KO MOVAS cells treated with rhCTHRC1. (E) Heatmap showing the expression levels of genes associated with extracellular matrix disassembly and contraction. (F) Immunoblotting analysis of ADAM9, α-SMA, SM22α, and p-ERK1/2 expression in MOVAS cells, knockouting Roas26 or Adam9 . (G) Immunoblotting analysis of ADAM9, α-SMA, SM22α, and p-ERK1/2 expression in MOVAS cells with or without treatment of ADAM9 inhibitor (ADAM9i). (H) H to I: Comprehensive profiles of genes in MOVAS cells with treatment of ADAM9i analyzed by bulk RNA-seq. Gene Ontology enrichment analysis of down-regulated genes in MOVAS cells treated with ADAM9i. (I) Heatmap showing the expression levels of metalloprotein genes in MOVAS MOVAS cells in the presence of rhCTHRC1cells treated with ADAM9i or DMSO. (J) Interaction of CTHRC1 with ADAM9 in human aortic smooth muscle cells (HASMCs) confirmed by Co-IP-blotting. (K) Western blotting analysis of ADAM9, α-SMA, SM22α, and p-ERK1/2 expression in HASMCs with treatment of ADAM9i. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Immunoprecipitation, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Incubation, Control, Expressing, Western Blot, RNA Sequencing

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Coomassie staining revealing the protein content in the samples coimmunoprecipitated with CTHRC1. Red box indicates the potential interactors. (B) The representative peptide mass spectrum of ADAM9 receptor on VSMCs. (C) Immunoblotting analysis of ADAM9 in MOVAS incubated with rhCTHRC1 for 0, 3, 6, 12, and 24 hours. (D) Violin plot of Adam9 gene expression in MOVAS cells with treatment of vehicle or rhCTHRC1. (E) Heatmap depicting the gene expression of ADAMs in control and AD mice. (F) F and G: transcriptome analysis of gene expression profiles in MOVAS cells without or with rhCTHRC1, knockouting Roas26 or Adam9 . A volcano plot of differentially expressed genes. The expression levels of Agt , Mmp2 and Mmp9 are down-regulated. (G) KEGG enrichment analysis of down-regulated genes in MOVAS cells in the presence of rhCTHRC1, knockouting Roas26 or Adam9 . (H) Relative mRNA levels of α-SMA and SM22α in MOVAS cells, without or with rhCTHRC1, knockouting Roas26 or Adam9 . (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.) (I) I to K: Comprehensive profiles of genes in MOVAS cells without or with treatment of ADAM9i by bulk RNA-seq. A volcano plot of differentially expressed genes. Egr1 , Mmp2 and Agt are marked with down-regulated expression. (J) KEGG enrichment analysis of down-regulated genes in MOVAS cells in the presence of rhCTHRC1, with or without ADAM9i. (K) Heatmap showing the expression levels of contraction-related genes in MOVAS cells without or with treatment of ADAM9i, incubated with rhCTHRC1.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Staining, Western Blot, Incubation, Gene Expression, Control, Transcriptome Wide Gene Expression, Expressing, Comparison, RNA Sequencing

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Amino acid sequence homology of immunogen with human or mouse CTHRC1 protein. (B) The affinity, specificity, and efficacy of anti-CTHRC1 Ab confirmed by ELISA (left) and immunoblotting (right) analysis. (C) C to E: mRNA sequencing results illustrating the transcriptomic profiles of aortas from WT mice treated with IgG Isotype and anti-CTHRC1 Ab (n=3 per group). PCA of transcriptome profiles in aortic tissues between the two groups. (D) Gene Ontology enrichment analysis of down-regulated genes between the two groups (anti-CTHRC1 Ab versus IgG Isotype). (E) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly. (F) F to K: WT female mice were intraperitoneally injected with anti-CTHRC1 Ab or IgG Isotype under saline or Ang-II/BAPN administration (n=10 per group). Kaplan-Meier survival rates analysis of the four groups of female mice. (Kaplan-Meier survival rates analysis, ns, no significance.) (G) Upper: Representative ultrasound images of aorta in female mice (scale bar, 1mm). Bottom: Measurements of maximum aortic diameter of mice (n=6 per group). (Mann Whitney test, *** P ≤ 0.001.) (H) Representative pictures of whole aortas in female mice treated with IgG Isotype or anti-CTHRC1 Ab. (I) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas in female mice. (Fisher’s exact test, * P ≤ 0.05.) (J) Representative images of histological staining with hematoxylin and eosin in aortic sections from the female mice. (K) Representative images of EVG staining from the female mice injected with anti-CTHRC1 Ab or IgG Isotype.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Injection, Saline, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) The sequences of heavy and light chains from anti-CTHRC1 monoclonal antibody (anti-CTHRC1 Ab), confirmed by variable region cloning, expressing and sequencing. (B) Molecular docking analysis showing the interaction between antibody-CTHRC1 antigen and CTHRC1-ADAM9 receptor at the molecular level. (C) Experiment design. WT mice were intraperitoneally injected with anti-CTHRC1 Ab or IgG Isotype at a dose of 5 μg/g body weight under saline or Ang-II/BAPN administration (n=10 per group). (D) Kaplan-Meier survival rates analysis of the mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration. (Kaplan-Meier survival rates analysis, * P ≤ 0.05, ns, no significance.) (E) Representative ultrasound images of aorta in mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration (scale bar, 1mm). (F) Measurements of maximum aortic diameter of mice in E (n=6-9 per group). (Student’s t-test, ** P ≤ 0.01.) (G) Representative pictures of whole aortas in mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration. The yellow arrows indicate the dissected sites of vascular lesions. (H) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas from the mice in E. (Mann-Whitney test, ** P ≤ 0.01.) (I) Representative images of histological staining with hematoxylin and eosin in aortic sections from the mice in E. Bottom: Measurements of aortic wall thickness of aortas (scale bar, 50 μm; scale bar, 200 μm). (Student’s t-test, **** P ≤ 0.00001.) (J) Representative images of Elastica van Gieson staining in aortic sections from the mice in E. Bottom: Numbers of elastin breaks per vessel. (scale bar, 50 μm; scale bar, 200 μm). (Student’s t-test, **** P ≤ 0.00001.) (K) K to O: scRNA-seq of the aortic tissues from WT mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration (n=2 per group). UMAP plot of the 42,623 single cells, displaying the identification of 8 major clusters. (L) The proportion of major cell types including SMCs, ECs and FBs in WT mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration (M) Bar diagram showing the percentage of 4 SMC subclusters in aortic SMCs of the two groups of mice. (N) Boxplot illustrating the differential expression of contractile genes in aortic SMCs of the two groups of mice. (*** P ≤ 0.001, ns, no significance.) (O) Gene Ontology enrichment analysis of differentially down-regulated genes in fibromyocytes of the two groups of mice. (P) Immunoblotting analysis and quantification of α-SMA and SM22α expression in aortas from the WT mice treated with IgG Isotype or anti-CTHRC1 Ab under Ang-II/BAPN infusion (n=6-9). (Student’s t-test, **** P ≤ 0.00001.) See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Cloning, Expressing, Sequencing, Injection, Saline, MANN-WHITNEY, Staining, Western Blot

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A to E: scRNA-seq of aortic tissues in healthy controls and human aortic dissection (n=2 per group). Experiment design of scRNA-seq. (B) Dotplot showing the expression level of CTHRC1 in fibroblasts of the aortas from AD patients and controls. Dash line in each group represents its mean expression value. (C) The proportion of 7 major cell clusters in AD patients and controls. (D) Boxplot displaying the differential gene expression of contractile genes in aortic SMCs of the controls and AD patients. (E) Gene Ontology enrichment analysis of differentially up-regulated genes in fibromyocytes from AD patients versus controls. (F) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly in aortic tissues from AD patients and controls by bulk RNA-seq. (*** P ≤ 0.001.) (G) Immunoblotting analysis of CTHRC1, ADAM9, p-ERK1/2 expression levels in human aortas (n=6). (H) Quantification of CTHRC1, ADAM9, p-ERK1/2 protein expression levels in G. (Student’s t-test, ** P ≤ 0.01, **** P ≤ 0.0001.) (I) Immunofluorescence staining of α-SMA and SM22α expression in human aortas (scale bar, 500 μm). (J) Laboratory tests for human serum CTHRC1, cTnT, and D-dimer contents in control individuals, acute myocardial infraction (AMI) and AD. (Student’s t-test, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns, no significance.) (K) Graphical abstract illustrating an important role of CTHRC1 acts as a candidate circulating biomarker and therapeutic target for aortic dissection through CTRHC1-ADAM9-ERK axis. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Dissection, Expressing, Gene Expression, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Control, Biomarker Assay

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A to E: scRNA-seq data of aortas from human healthy controls and AD patients (n=2 per group). Violin plots showing data characteristics on nFeature, nCount and percent.mt after quality control of the two groups. (B) Violin plot showing the expression level of the housekeeping gene beta-actin (ACTB) of the four samples. The relative stability of ACTB expression indicates consistency. (C) UMAP plot of 45,501 single cells from 4 samples colored according to 7 different cell clusters. (D) UMAP plot of SMCs colored according to the identified 4 SMC subclusters. (E) Bar graphs depicting the numbers of differentially expressed genes in SMC subclusters in healthy controls and AD patients. (F) Gene Ontology enrichment analysis of down-regulated genes in contractile SMCs of the aortas. (G) KEGG enrichment analysis of differentially expressed genes in aortas between the two group. (H) Representative imaging results of contrast-enhanced computed tomography (CT) scanning of patients with aortic dissection, coronary angiography (CAG) of patients with acute myocardial infarction (AMI). The yellow and blue arrows indicate lesion sites in aortas and coronary arteries. (I) Clinical tests for serum biomarkers (Myoglobin, CK-MB, hsCRP and BNP) between AMI or AD patients. (Student’s t-test, * P ≤ 0.05, ** P ≤ 0.01, *** P≤ 0.001, ns, no significance.) (J) A corresponding ELISA kit developed by our team for detecting serum CTHRC1 levels. (K) CTHRC1 point-of-care testing (POCT) equipment, providing rapid and reliable results from a single drop of in less than 10 minutes. (L) The process of assessing chest pain during ambulance transport or in the ER.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Control, Expressing, Imaging, Computed Tomography, Dissection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) can inhibit the stimulation of cluster of differentiation 4 positive T (CD4 + T) cells on microglia activation in collagen-induced arthritis (CIA). (A) The expression level of CD4 was conducted by Western blot (left) and blot quantification and statistics (right) ( n = 3 per group). (B) Transwell assay showed the cell number across the upper chamber in CD4 + T cells from different groups ( n = 15 per group). (C) The double-immunofluorescence staining of ionized calcium-binding adapter molecule 1 (IBA1) and CD4 ( n = 3 per group). (D) A schematic illustration of the microglia intervened with supernatants of CD4 + T cells derived from different groups of mice. (E) Representative pictures of immunofluorescence staining and the relative fluorescence for IBA1, CD68, and CD40 of the microglia intervened with supernatants of CD4 + T cells in different groups of mice ( n = 3 per group). (F) The level of interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) conducted by enzyme-linked immunosorbent assay (ELISA) ( n = 6 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control or control CD4 + T cell group; # P < 0.05, compared with CIA or CIA/rheumatoid arthritis (RA) CD4 + T cell group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Activation Assay, Expressing, Western Blot, Transwell Assay, Double Immunofluorescence Staining, Binding Assay, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) inhibits T helper 17 (Th17) differentiation and spinal infiltration. (A) Volcano plot of differentially expressed genes (DEGs) between cluster of differentiation 4 positive T (CD4 + T) cells of control and collagen-induced arthritis (CIA) group. (B) Volcano plot of DEGs between CD4 + T cells of CIA mice with or without LUT treatment. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated genes in CIA CD4 + T cells. (D) KEGG pathway enrichment analysis of downregulated genes in CIA + LUT CD4 + T cell. (E) Representative flow cytometry plots of spleen CD4 + interleukin-17 (IL-17) + T cells measured as CD4 + IL-17A + cell percentages (left), and the percentage of spleen CD4 + IL-17A + T cell was calculated by CytExpert 2.4.0.28 ( n = 3 per group) (right). (F) Enzyme-linked immunosorbent assay (ELISA) was employed to quantify IL-17A and IL-22 concentrations in spinal dorsal horn (SDH) samples collected from mice in each experimental group ( n = 6 per group). (G) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to quantify IL-17 a and IL-22 messenger ribonucleic acid (mRNA) in SDH samples collected from mice in each experimental group ( n = 6 per group). (H) The Venn diagram showed the intersection of the genes upregulated in CIA mice, genes downregulated after LUT treatment, and genes enriched in the Th17 differentiation pathway. (I) The heat map showed the mRNA expression profile of the overlapping genes ( n = 3 per group). (J) qRT-PCR validation of mRNA expression of the overlapping genes (nuclear factor of activated T cells 2 ( Nfatc2 ) , IL23α , IL1 receptor accessory protein ( IL1rap ), signal transducer and activator of transcription 5A ( Stat5a ), and zeta chain of T cell receptor associated protein kinase 70 ( Zap70 )) ( n = 4 per group). (K) The immunofluorescence intensity of NFATC2 in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (L) The double-immunofluorescence staining was used to verify the level of co-localization of NFATC2 and CD4 in SDH ( n = 4 per group). The insets and white arrows indicate representative images of NFATC2-CD4 co-localization and cells exhibiting NFATC2-CD4 co-localization, respectively. ∗ P < 0.05 and ∗∗ P < 0.01, compared with control group; # P < 0.05 vs. CIA or rheumatoid arthritis (RA) group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. CIA + LUT-H: CIA + LUT high dose; HIF-1: hypoxia inducible factor-1; NF-κB: nuclear factor kappa B; FoxO: forkhead box O; VEGF: vascular endothelial growth factor; PE-A: phycoerythrin-area; APC-A: allophycocyanin-area; UL: upper left; UR: upper right; LL: lower left; LR: lower right; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Biomarker Discovery, Immunofluorescence, Double Immunofluorescence Staining

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) inhibited nuclear factor of activated T cells 2 (NFATC2)-drived T helper 17 (Th17) differentiation and spinal infiltration in collagen-induced arthritis (CIA) model. (A) Representative flow cytometry plots of cluster of differentiation 4 (CD4) + interleukin-17 (IL-17) + T cells measured as IL-17A + CD4 + cell percentages (left), and the percentage of spleen Th17 cells was calculated by CytExpert 2.4.0.28 ( n = 3 per group) (right). (B) Quantitative live cell tracking of motility reveals significant increases in migration speed and distance of Th17 polarization cytokine cocktail-stimulated CD4 + T cells by transfected with NFATC2 short hairpin RNA (shRNA) plasmid in vitro (top). Visual narrative of cell morphology through time-lapse imaging and tracking (middle). Distinct cellular structures such as lamellipodia and uropodia are annotated with red arrow ( n = 20 per group) (bottom). (C) The double-immunofluorescence staining was used to verify the level of co-localization of IL-17A and CD4 in spinal dorsal horn (SDH) ( n = 4 per group). The arrows indicate IL-17A + CD4 + cells. The inset provides a detailed view of cells where IL-17A and CD4 are co-localized. (D) The Spearman correlation analysis of the percentage of IL-17A + CD4 + T cells and the expression level of NFATC2 ( n = 6). (E–G) The immunofluorescence staining and Western blot were used to verify the level of IL-17A (E), phosphorylated signal transducer and activator of transcription 3 (pSTAT3) (F), and ionized calcium-binding adapter molecule 1 (IBA1) (G) in SDH ( n = 3–4 per group). (H) Mechanical pain threshold and thermal withdrawal latency in different groups ( n = 8 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with scramble shRNA, control, or CD4 c yclization recombination enzyme (Cre) + scramble shRNA groups; # P < 0.05 and ## P < 0.01, compared with CIA, or CD4 Cre + scramble shRNA + CIA group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) or two-way ANOVA followed by post hoc Dunnett's multiple comparisons test. SSC-A: side scatter-area; PE-A: phycoerythrin-area; DAPI: 4′,6-diamidino-2-phenylindole; CIA + LUT-H: CIA + LUT high dose; mRNA: messenger RNA; AAV: adeno-associated virus.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Flow Cytometry, Cell Tracking Assay, Migration, Transfection, shRNA, Plasmid Preparation, In Vitro, Imaging, Double Immunofluorescence Staining, Expressing, Immunofluorescence, Staining, Western Blot, Binding Assay, Control, Two Tailed Test, Virus

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Nuclear factor of activated T cells 2 (NFATC2) orchestrates T helper 17 (Th17) pathogenicity via a protein kinase C epsilon (PRKCE)-signal transducer and activator of transcription 3 (STAT3) cascade. (A) The binding density of NFATC2 was visualized by deepTools: the heatmap presents the cleavage under targets and tagmentation (CUT&Tag) counts on the different NFATC2 binding peaks in cluster of differentiation 4 positive T (CD4 + T) cells derived from control and collagen-induced arthritis (CIA) mice ( n = 3 per group). (B) Genome-wide distribution of upregulated and downregulated NFATC2-binding peaks in CD4 + T cells derived from control and CIA mice. (C) Gene Ontology biological process (GO-BP) analysis of the NFATC2 binding peaks at promoter genes. (D) Genome browser tracks of CUT&Tag signal at the representative target gene loci ( n = 3 per group). (E) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (ChIP-qPCR) assay detected NFATC2 levels at the PRKCE promoter regions in CD4 + T cells derived from control and CIA mice ( n = 3 per group). (F) The co-localization of PRKCE and pSTAT3 was detected by laser confocal microscopy ( n = 3 per group). (G) The levels of interleukin-17A (IL-17A) and retinoic acid-related orphan receptor gamma t (RORγt) were detected by enzyme-linked immunosorbent assay (ELISA) ( n = 6 per group). (H, I) Transwell assay (H) and quantification (I) showed the cell number across the upper chamber in cells treated with scramble short hairpin RNA (shRNA) or NFATC2 shRNA ( n = 5 per group). (J) The co-localization of C–C chemokine receptor type 6 (CCR6) and C–C motif chemokine ligand 20 (CCL20) was detected by laser confocal microscopy ( n = 3 per group). (K) The levels of CCR6 and CCL20 were detected by ELISA ( n = 6 per group). (L) The levels of IL-17A and RORγt were detected by ELISA under different interventions ( n = 6 per group). (M, N) Transwell assay (M) and quantification (N) showed the cell number across the upper chamber in cells under different interventions ( n = 5 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with scramble shRNA or control group; # P < 0.05, compared with NFATC2 shRNA group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. TSS: transcription start site; TTS: transcription termination site; bp: base pairs; Ifna11 : interferon alpha 11; Msh2 : mutator S (MutS) homolog 2; Psen2 : presenilin 2; Sanbr : switch 3 (SWI3), alteration/deficiency in activation 2 (ADA2), nuclear receptor co-repressor (N-CoR), transcription factor III B (TFIIIB) and bric-a-brac, tramtrack, broad complex domain regulator of class switch recombination (CSR); Tmem98 : transmembrane protein 98; DAPI: 4′,6-diamidino-2-phenylindole; OE: overexpression.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Binding Assay, Derivative Assay, Control, Genome Wide, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Transwell Assay, shRNA, Two Tailed Test, Activation Assay, Over Expression

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Effects of luteolin (LUT) treatment on nuclear factor of activated T cells 2 (NFATC2) transcription and lactic acid levels. (A, B) The total ion chromatogram of metabolites between cluster of differentiation 4 positive T (CD4 + T) cells derived from collagen-induced arthritis (CIA) mice with or without LUT (50 mg/kg) treatment: positive (A) and negative (B) ion models. (C, D) The principal component analysis (PCA) score plot of CD4 + T cells. It represents samples in the groups were closely cluster to one another: positive (C) and negative (D) ion models ( n = 3 per group). (E) Heatmap of differential metabolites derived from CIA mice with or without LUT treatment. The upregulated metabolites were marked in red, and the downregulated metabolites levels were presented in blue ( n = 3 per group). (F) Bubble map of the impact values of metabolic pathways, where the horizontal coordinate is the impact values enriched into different metabolic pathways, and the vertical coordinate is the enrichment pathway. The size of the dots indicates the corresponding number of metabolites on the pathway. The color of the dot reflects the P value, where the redder the color, the smaller the P value, and the bluer the color, the larger the P value. (G) The lactate level of CD4 + T cells derived from CIA mice treated with LUT or not based on global untargeted metabolomics ( n = 3 per group). (H) The lactate level in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (I) Lactate content assay kit and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to detect the lactate level and Nfatc2 expression in T helper 17 (Th17) polarization cytokine cocktail-induced CD4 + T cells at different LUT concentrations (10, 20, and 40 μM) ( n = 6 per group). (J) The correlation of the lactate and the expression level of Nfatc2 in Th17 polarization cytokine cocktail-induced CD4 + T cells at 40 μM LUT concentrations ( n = 6). ∗ P < 0.05, compared with control CD4 + T cell group; # P < 0.05, compared with rheumatoid arthritis (RA) CD4 + T cell group; && P < 0.01, compared with CIA CD4 + T cell group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. PC: principal component; HIF-1: hypoxia-inducible factor-1; mTOR: mammalian target of rapamycin.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Derivative Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Control, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) could suppress nuclear factor of activated T cells 2 (NFATC2) transcription by decreasing histone H3 lysine 9 lactylation (H3K9la) in cluster of differentiation 4 positive T (CD4 + T) cells. (A) Western blot analysis showed the level of global lactylation in spleen CD4 + T cells of collagen-induced arthritis (CIA) mice with or without LUT treatment ( n = 5 per group). (B) The immunofluorescence staining was used to verify the localization of global lactylation and 4′,6-diamidino-2-phenylindole (DAPI) in CD4 + T cells. The white arrow shows the nucleus. (C–E) The Western blot was used to verify the expression of H3K9la (C), histone h3 lysine 18 lactylation (H3K18la) (D), and histone h3 lysine 23 lactylation (H3K23la) (E) in spleen CD4 + T cells of CIA mice with or without LUT treatment ( n = 4 per group). (F) The immunofluorescence intensity of H3K9la in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (G) Chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (ChIP-qPCR) detected the H3K9la level at the NFATC2 promoter regions in CD4 + T cells with or without sodium lactate (NaLa) treatment ( n = 3 per group). (H) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays monitoring expression of the NFATC2 in CD4 + T cells treated with different concentrations of NaLa (0, 5, 10, and 25 mM) for 24 h ( n = 6 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control CD4 + T cell group; # P < 0.05, compared with CIA or rheumatoid arthritis (RA) CD4 + T cell group, by two-tailed Student's t -test or repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's or Tukey multiple comparisons test. Pan Kla: pan-lysine lactylation.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Luteolin attenuates RA-associated chronic pain by targeting the LDHA/H3K9la/NFATC2 axis to suppress Th17 cell differentiation and central infiltration

doi: 10.1016/j.jpha.2025.101373

Figure Lengend Snippet: Luteolin (LUT) could inhibit the expression and activity of lactate dehydrogenase A (LDHA). (A) Venn gram showed the intersection of upregulated genes in spleen cluster of differentiation 4 positive T (CD4 + T) cells of collagen-induced arthritis (CIA) mice and downregulated genes in spleen CD4 + T cells of CIA mice with LUT treatment and the genes enriched in the glycolytic pathway according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. (B) Heat map of differential shows the messenger ribonucleic acid (mRNA) level of hexokinase domain containing 1 ( Hkdc1 ) , phosphoglycerate kinase 1 ( Pgk1 ), Ldha , pyruvate kinase m1/2 ( Pkm ), bisphosphoglycerate mutase ( Bpgm ), phosphofructokinase platelet ( Pfkp ), phosphoglucomutase 1 ( Pgm1 ), and glucose-6-phosphate isomerase 1 ( Gpi1 ) in CD4 + T cells of CIA mice with or without LUT administration ( n = 3 per group). (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) monitors the mRNA expression of Hkdc1 , Pgk1 , Ldha , Bpgm , Pkm , Pfkp , Pgm1 , and Gpi1 in CD4 + T cells of CIA mice with or without LUT administration ( n = 4 per group). The dashed lines serves to highlight the genes that were significantly upregulated in CD4 + T cells of the CIA group and significantly downregulated upon LUT treatment, thereby drawing clearer attention to the key findings. (D) The immunofluorescence intensity of LDHA in peripheral CD4 + T cells from each group was detected ( n = 15 per group). (E–H) A model of LUT binding with LDHA protein generated by molecular docking: surface diagram of LUT and LDHA binding (E), enlarged view of the surface diagram of LUT and LDHA binding (F), three-dimensional (3D) diagram of the interaction between LUT and LDHA (G), and 2D diagram of the interaction between LUT and LDHA (H). (I–K) The surface plasmon resonance (SPR) analysis of LUT-LDHA interaction (1.9–62.5 μmol/L): steady-state affinity fitting curve, showing the binding signal as a function of analyte concentration (I), kinetic fitting using a 1:1 binding model, where the affinity is calculated from the ratio of association to dissociation rates (J), and sensogram depicting real-time binding responses, with data points recorded every 0.1 s (K). (L) The root mean square deviation (RMSD) analysis of 100 ns-length molecular dynamics (MD) simulation results for LDHA and LUT. (M) Radius of gyration (Rg) graph of LDHA docked with LUT from 0 to 100 ns time scale. (N) The 3D free energy landscape of LDHA docked with LUT. (O) The 2D free energy landscape of LDHA docked with LUT. (P) The average number of hydrogen bonds between each ligand throughout 100 ns simulations. (Q) LDHA activity was measured in CD4 + T cells derived from control, CIA, and CIA + LUT mice ( n = 6 per group). (R) LDHA activity was measured in CD4 + T cells derived from different group ( n = 15 per group). ∗ P < 0.05 and ∗∗ P < 0.01, compared with control CD4 + T cell group; # P < 0.05 and ## P < 0.05, compared with CIA or rheumatoid arthritis (RA) CD4 + T cell group, by repeated-measures one-way analysis of variance (ANOVA) followed by post hoc Dunnett's multiple comparisons test. CIA + LUT-H: CIA + LUT high dose; DAPI: 4′,6-diamidino-2-phenylindole; K D : dissociation constant.

Article Snippet: The following reagents and materials were used in this study: immunization grade bovine type II collagen (Cat. No.: 20021; Chondrex, Inc.), LUT (Cat. No.: B20888 ; Shanghaiyuanye Bio-Technology Co., Ltd., Shanghai, China), anti-CD4 antibody (GK 1.5) (Cat. No.: 16-0041-86; Thermo Fisher Scientific Inc., Waltham, MA, USA), recombinant mouse C–X–C motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 alpha (SDF-1α) protein (Cat. No.: 460-SD-050; R&D Systems, Minneapolis, MN, USA), ultra-low endotoxin, azide-free (LEAF)TM purified anti-mouse CD3 antibody (Cat. No.: 100238; BioLegend, San Diego, CA, USA), ultra-LEAFTM purified anti-mouse CD28 antibody (Cat. No.: 102116; BioLegend), IL-1β (Cat. No.: 211-11B; PeproTech, Rocky Hill, NJ, USA), IL-23 (Cat. No.: 589002; BioLegend), IL-6 (Cat. No.: 216-16; PeproTech), transforming growth factor beta (TGF-β) (Cat. No.: 594509; BioLegend), fluorescein isothiocyanate (FITC)-CD3 (Cat. No.: 100203; BioLegend), allophycocyanin (APC)-CD4 (Cat. No.: 100515; BioLegend), phycoerythrin (PE)-IL-17A (Cat. No.: 506903; BioLegend), magnetic activated cell sorting (MACS) buffer (Cat. No.: 130-091-221; Miltenyi Biotec, Bergisch Gladbach, Germany), CD4 + T Cell Isolation Kit (mouse) (Cat. No.: 130-104-454; Miltenyi Biotec), CD4 + T Cell Isolation Kit (human) (Cat. No.: 130-096-533; Miltenyi Biotec), sodium l -lactate (Cat. No.: 71718; Sigma-Aldrich, St. Louis, MO, USA), Lactic Acid Content Assay Kit (Cat. No.: BC2230; Solarbio, Beijing, China), lactate dehydrogenase (LDH) Activity Assay Kit (Cat. No.: A020-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), mouse IL-1β, IL-6, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits (Cat.

Techniques: Expressing, Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Binding Assay, Generated, SPR Assay, Concentration Assay, Derivative Assay, Control

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: mRNA expression analysis in Nf1 mutant mouse Schwann cells. (A) Microarray analysis was used to compare genome-wide expression levels between normal mouse Schwann cells and Nf1 mutant Schwann cells. The control for each comparison was Cy3-labeled cDNA generated from normal mouse Schwann cell mRNA. For each of four Nf1 mutant Schwann cell samples (Nf1+/−, Nf1−/−, Nf1−/− TXF, and Nf1−/− TXF treated with FTI), mRNA was used as a template to synthesize Cy5-labeled cDNA. Cy3- and Cy5-labeled cDNA probes were hybridized simultaneously to the Incyte Genomics MouseGEM 1.0 cDNA microarray. Relative intensities of Cy3 versus Cy5 fluorescent signals for each cDNA target sequence were analyzed with GeneSpring software. The most changes were observed in the Nf1−/− TXF cells (genes upregulated in Nf1−/− TXF are red; genes downregulated in Nf1−/− TXF are green). Expression of one target cDNA, BLBP (black line), was 26-fold above normal in the Nf1−/− TXF cells and not normalized by FTI treatment. (B) RT-PCR analysis confirmedthe microarray result of elevated BLBP expression in Nf1−/− TXF cells. Reverse transcriptase (RT) was omitted from duplicate samples to control for DNA contamination. Primers for BLBP (∼200-bp amplicon) and actin control primers (∼500-bp amplicon) were included in the mixture for each 40-cycle reaction. The plasmid positive control for BLBP amplification is the UniGEM clone (Incyte Genomics) containing the BLBP cDNA insert spotted on the microarray. (C) Quantitative real-time PCR of BLBP normalized to GAPDH resulted in a 145-fold change over expression in Nf1−/− TXF cells compared to wild-type mouse Schwann cells. Rn, fluorescent signal intensity; horizontal starred line, chosen threshold at geometric phase of amplification.

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Expressing, Mutagenesis, Microarray, Genome Wide, Labeling, Generated, Sequencing, Software, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Positive Control, Real-time Polymerase Chain Reaction, Over Expression

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: Mouse neuron-Schwann cell coculture. Anti-BLBP promotes extension of Nf1−/− TXF cell processes along axons. Wild-type (A and B) or Nf1−/− TXF (C and D) mouse Schwann cells labeled with Cell Tracker green were preincubated with rabbit IgG (A and C) or anti-BLBP antibodies (B and D) and seeded onto DRGN cultures stripped of endogenous Schwann cells. Two days after seeding, cocultures were fixed and stained with antineurofilament antibodies followed by Cy3 (red)-conjugated secondary antibodies. Confocal images obtained with Zeiss LSM Image Browser software are shown. Single cells are representative of the majority observed with each treatment. Arrowheads indicate Schwann cell processes. The asterisk indicates the region which is magnified fivefold in the inset. The scale bar in panel C equals 5 μm and also applies to panels A, B, and D. (E) Lower magnification (scale bar, 5 μm) of Nf1−/− TXF on DRGN cultures in the presence of anti-BLBP antibodies. Arrowheads indicate processes from two cells extending along neurites; other cells lack processes. (F) Extension of Nf1−/− TXF cell processes in the presence of anti-BLBP antibodies is statistically significant. The percentages of Nf1−/− TXF cells extending processes along axons in the presence of control IgG (gray bar) or anti-BLBP antibodies (black bar) are graphed. Error bars reflect standard deviations in a Student t test (P = 0.003).

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Labeling, Staining, Software

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: Interactome of MALAT1 lncRNA by RAT-seq. A. RAT-seq assay. MALAT1 lncRNA was in situ reverse transcribed using MALAT1-specific complementary primers at 60°C with biotin-dCTP. The biotin-MALAT1 cDNA chromatin complex was isolated by streptavidin beads and cDNAs were isolated for Illumina library sequencing. RAT-seq will generate a genome-wide target interaction network for MALAT1 lncRNA in breast cancer cells. B. Gene ontology enrichment pathway analysis of the MALAT1 RAT-Seq data. GO enrichment was analyzed with Cytoscape software. C. The MALAT1 RAT-seq interactome. The MALAT1 interactome was drawn based on the enrichment fold of the top RAT-Seq pathway target genes.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: In Situ, Reverse Transcription, Isolation, Sequencing, Genome Wide, Software

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 binds to the EEF1A1 promoter and epigenetically regulates its activity. A. The RAT-seq IGV binding of MALAT1 lncRNA at the EEF1A1 locus. MALAT1-RAT: the RAT-seq library created by the MALAT1-specific complementary primers; RC-RAT: the RAT-seq control library created by random oligonucleotide primers; pEEF1A1: EEF1A1 promoter; 3’-CT, 5’-CT: the 3’- and 5’-control sites; E1-E8: EEF1A1 exons. B. Quantitation of EEF1A1 binding in the MALAT1-specific RAT-seq products and the negative control RAT-seq products. C. EEF1A1 expression levels by Q-PCR in MALAT1-knockdown cells. β-Actin was used as an internal control. **P < 0.01 as compared with the control groups. D. Western blot of eEF1A1. Note the reduced expression of eEF1A1 in MALAT1-knockdown breast cancer cells. GAPDH was used as control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Activity Assay, Binding Assay, Control, Quantitation Assay, Negative Control, Expressing, Knockdown, Western Blot

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 epigenetically regulates EF1A1. A. pEEF1A1-luciferase assay. The EEF1A1 promoter (pEEF1A1) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with pGL3-Basic vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. **P < 0.01 as compared with the control groups. B. Quantitation of Histone 3-K4 (H3K4) trimethylation. All data are presented as the relative values after normalization over the input DNA. *P < 0.05 as compared with control.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Luciferase, Sequencing, Clone Assay, Reporter Assay, Plasmid Preparation, Negative Control, Control, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 is dysregulated in breast cancer. A. Unsupervised hierarchical clustering analysis of the significantly differentially expressed genes in paracancer tissues and tumor tissues. Data from 64 mammary tissues was downloaded from TCGA database. In the heatmap, the normalized expression values are represented in shades of green and red, indicating the expression being above and below the median expression value across the samples. B. The normalized expression level of MALAT1 in 64 mammary tissues (32 paracancer tissues and 32 tumor tissues). ***P < 0.01 as compared with the paracancer group. C. Q-PCR quantitation of MALAT1 expression in breast cancer cell lines.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Expressing, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: The role of MALAT1 in cell proliferation and cell cycle. A. MALAT1 shRNA knockdown in two breast cancer cell lines. MALAT1 expression was examined by Q-PCR in CTL (non-transfected control), shNC (random shRNA non-targeting control), shMALAT1 (MALAT1 shRNA-1 transfected cells), and shMALAT1-2 (MALAT1 shRNA-2 transfected cells). β-Actin was used as an internal control. **P < 0.01 as compared with CTL and shNC controls. B. Cell Proliferation. CCK-8 assay was used to determine cell growth viability at 0, 24, 48, 72 and 96 hour time points. C. Cell cycle. Flow cytometry was used to measure cell cycle profile with propidium iodide staining. Cell numbers were counted according to DNA content of G0/G1, S and G2/M phases. The statistical results are shown on the right panel. *P < 0.05 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: shRNA, Knockdown, Expressing, Transfection, Control, CCK-8 Assay, Flow Cytometry, Staining

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: MALAT1 knockdown inhibits cell invasion in breast cancer cells. Representative images of invading MDA-MB231 cells (A) and SKBR3 cells (B) are showed on the left panel. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. Quantitation of invaded cells is shown in the right panel, mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, shRNA, Control, Transfection, Quantitation Assay

Journal: American Journal of Cancer Research

Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

doi:

Figure Lengend Snippet: EEF1A1 rescues the effect induced by MALAT1 knockdown. A. Overexpression of EEF1A1 in breast cancer cells. The expression of EEF1A1 was quantitated by Q-PCR. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. β-Actin was used as an internal control. ***P < 0.001 as compared with the control groups. Overexpression of eEF1A1 in breast cancer cells was measured by Western blot. B. Cell growth viability as measured by CCK-8 assay. C. Cell invasion as examined by Transwell assay. Quantitation of invaded cells was shown as mean ± SD, **P < 0.01 as compared with the control groups.

Article Snippet: The biotinylated- MALAT1 -cDNA/chromatin DNA complex was pulled down with biotin-streptavidin magic beads (Invitrogen, CA).

Techniques: Knockdown, Over Expression, Expressing, shRNA, Control, Transfection, Western Blot, CCK-8 Assay, Transwell Assay, Quantitation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 2. Characteristics of the HHR thymus and differentiation status of thymocytes in the thymus. (A) Graphs show the weights and cellularities of SDR and HHR thymi at 4–5 wk of age. Three SDRs and HHRs were used for each analysis. Data are shown as mean 6 SD. *p , 0.05. (B) Histological sections of thymi from 4-wk-old SDRs and HHRs were stained with H&E. Scale bars represent 1 mm. Representative results of two SDRs and HHRs are shown. (C) The differentiation status of thymocytes was analyzed with a flow cytometer. Representative results of three SDRs and HHRs are shown. An arrow indicates a population of DP thymocytes with decreased CD4 levels. Averaged values from three independent flow cytometric analyses for the proportions of DP, CD4-SP, CD8-SP, and DN thymocytes are shown in the lower graph. Data are shown as mean 6 SD. (D) Expression levels of Cd4 and Cd8 genes in the thymus were analyzed by real-time RT-PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Staining, Cytometry, Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 3. nTreg numbers in the HHR thymus. (A) Expression levels of Cd25 and Foxp3 genes in CD4-SP thymocytes were analyzed by real-time RT- PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) Upper, The proportion of CD4+CD25+ cells in the thymus was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of CD4+CD25+ cells are shown in the graph. Data are shown as mean 6 SD. Lower, The proportion of Foxp3+ cells in the CD4+CD25+ cell fraction was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of Foxp3+ cells in the CD4+CD25+ cell fraction are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. (C) Upper, Estimated values for the proportion of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of Foxp3+ cells in the CD4+CD25+ cell fraction obtained from flow cytometric analysis and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. Lower, Estimated values for the absolute number of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of total thymus cell number (Fig. 2A) and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 5. Loss of Ly49s3 gene expression in HHR thymic cDCs. (A) Left, Genome-wide microarray CGH analysis, performed with genomic DNA from SDR and HHR livers, shows the deletion of four Ly49 family genes—Ly49s4, Ly49i4, Ly49s3, and Ly49i3—in chromosome 4 at the q42 region (shaded area). Data shown are representative of two independent analyses. Right, Genomic PCR with DNA from SDR and HHR livers was performed to confirm the deletion of DNA in this region. As an internal standard, the Ccr4 gene, located at chromosome 8q32, was used. (B) Left, RT-PCR analysis of the expression of the Ly49s3 gene was performed with total RNA from SDR and HHR thymi. As an internal standard, the Gapdh gene was used. Middle, RT- PCR analysis of Ly49s3 gene expression in DP, CD4-SP, and CD8-SP thymocytes of the SDR thymus was performed with total RNA from the cells. Note that it was not possible to isolate pure DN thymocytes by positive and/or negative selection using CD4 and CD8a microbeads because the remaining cells after the selection of DP, CD4-SP, and CD8-SP cells are a mixture of DN thymocytes and all of the other types of cells. Right, RT-PCR analysis of Ly49s3 gene expression in cDCs of SDR and HHR thymi was performed with total RNA from the cells.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Gene Expression, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Selection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 6. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture. (A) A total of 1 3 106 CD4-SP thymocytes isolated from the SDR thymus were cultured with 2.5 3 105 cDCs from the SDR thymus, and 1 3 106 CD4-SP thymocytes from the HHR thymus were cultured with 2.5 3 105 cDCs from the HHR thymus. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) To confirm the expression levels of Foxp3 and CD25, an additional mixed-cell culture experiment was performed, and the proportion of Foxp3+ or CD25+ cells was determined by flow cytometric analysis. (C) As control experiments, 1 3 106 CD4-SP thymocytes were cultured alone for 3 d and the same real-time RT-PCR analyses as above were performed. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for SDR cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (D) Effects of cDCs on nTreg marker gene expression. The expression levels of nTreg marker genes in mixed-cell (A) and control cultures (C) are shown in the same graph relative to the value for the SDR control culture, which is set at 1. Statistical analysis was performed between the induction ratios of gene expression. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 1 3 106 CD4+CD82CD252 thymocytes isolated from SDR and HHR thymi were cultured with 2.5 3 105 cDCs isolated from SDR and HHR thymi, respectively. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Isolation, Quantitative RT-PCR, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 7. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture using Ly49s3-expressing HHR thymic cDCs. (A) The FLAG- tagged Ly49s3 structure is schematically represented. R: arginine residue. (B) Left, 293T cells were transfected with recombinant vectors before being packaged into the virus, and cell lysates were subjected to Western blot analysis with the anti-FLAG M2 Ab. Right, The proteins on the membrane were stained with fast green. (C) Left, cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3, and the expression of the fusion protein on the surface of cDCs was confirmed by fluorescence microscopic observations with the anti-FLAG M2 Ab. Right, Phase contrast appearance of the identical cells shown in the left photographs. Note dendrites on the surface of the cells. The cells were suspended in buffer and all the photos were taken. Original magnification 3800. (D) A total of 2.5 3 105 cDCs isolated from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 (Ly) or the mock vector (Mo) and then mixed with 1 3 106 CD4-SP thymocytes isolated from the HHR thymus. At 3 d later, total RNAwas extracted and the expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture, using cDCs transduced with the mock vector, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 2.5 3 105 cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 and then mixed with 1 3 106 CD4-SP thymocytes from the HHR thymus. Next 5 mg of anti-rat MHC class I Ab or normal IgG was added to the culture. At 3 d later, total RNA was extracted, and the expression levels of nTreg marker genes and MHC class II genes were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture in the presence of normal IgG, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (F) Summary of the experiments performed using the lentiviral vector of the Ly49s3 gene and anti-MHC class I Ab. The level of each gene is shown relative to the value for mock-transduced cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Residue, Transfection, Recombinant, Virus, Western Blot, Membrane, Staining, Transduction, Plasmid Preparation, Isolation, Quantitative RT-PCR

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: cAMP signaling induces CRTC2 nuclear translocation and condensate formation. a) Live‐cell imaging of ectopically expressed CRTC2‐eGFP, CREB‐eGFP, CREM‐eGFP, and ATF1‐eGFP in 293T cells without (DMSO) and with forskolin (FSK) treatment (upper). Quantification of condensates number per nucleus (lower). b) In vitro droplet formation assay with recombinant CRTC2‐IDR‐eGFP at different protein concentrations (left). Quantification of the size of droplets (right). c) Representative images of the in vitro FRAP experiment with recombinant CRTC2‐IDR‐eGFP (upper). Quantification of FRAP data for CRTC2‐IDR‐eGFP puncta (lower). d) In vitro droplet formation assay of recombinant eGFP fusion proteins fused with wild‐type (WT) CRTC2‐IDR or CRTC2‐IDR mutants (left). Quantification of the size of droplets (right). e) Live‐cell imaging of ectopically expressed CRTC2‐eGFP in 293T cells. Arrows indicate representative CRTC2 puncta that fused over time. The dotted line area indicates the nucleus. f) Representative images of the FRAP experiment with ectopically expressed CRTC2‐eGFP in 293T cells (upper). The dotted line area indicates the nucleus. Quantification of FRAP data for CRTC2‐eGFP puncta (lower). g) Live‐cell images of ectopically expressed WT CRTC2‐eGFP or CRTC2‐IDR‐R>A mutant (R>A‐eGFP) in 293T cells (left). Quantification of cells with eGFP foci and western blot analysis of CRTC2‐eGFP or CRTC2‐IDR‐R>A expression (right). h) Live‐cell snapshots of ectopically expressed mCherry‐CRY2 fusion proteins fused with WT CRTC2‐IDR (upper) or CRTC2‐IDR‐R>A mutant (lower) in 293T cells before and after blue light stimulation (left). Quantification of cells with mCherry foci before and after blue light stimulation (right). Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. n.s., not significant. Scale bar, 5 µm (a), 10 µm (b,d). All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Translocation Assay, Live Cell Imaging, In Vitro, Tube Formation Assay, Recombinant, Mutagenesis, Western Blot, Expressing

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: P‐TEFb interacts with CRTC2 and enhances CRTC2‐dependent transcription. a) Immunofluorescence analysis of the co‐localization of CRTC2 and CycT1 in HeLa cells treated with DMSO or FSK (left). Line scans along the dotted lines in the images (right). b) Co‐IP assay examining the interaction between endogenous CRTC2 and P‐TEFb in 293T cells treated with DMSO or FSK for 1 h. c) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells treated with the indicated concentration of FSK or H89. d) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 in 293T cells ectopically expressing Myc‐SIK1. e) Co‐IP assay examining the interactions between FLAG‐CDK9 and HA‐CRTC2 (WT or S171/274A mutants) in 293T cells. f) Quantification of CRE‐luc luciferase activity in 293T cells transfected with the indicated amounts of FLAG‐CDK9 plasmids and treated with DMSO or FSK (10 µ m ) for 6 h. g) Quantification of CRE‐luc luciferase activity in 293T cells treated with flavopiridol (FP, 300 n m ) and FSK (10 µ m ) for 6 h. h) Quantification of CRE‐luc luciferase activity in HA‐CRTC2‐expressing 293T cells infected with lentivirus carrying sgRNA against CDK9 (sgCDK9) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. i) Quantification of CRE‐luc luciferase activity in FLAG‐CDK9‐expressing 293T cells infected with lentivirus carrying sgRNA against CRTC2 (sgCRTC2) or GFP (sgControl) and treated with DMSO or FSK (10 µ m ) for 6 h. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001; n.s., not significant. All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Concentration Assay, Expressing, Luciferase, Activity Assay, Transfection, Infection

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 extracts P‐TEFb from HEXIM1‐containing inhibitory complex. a) Live‐cell imaging of 293T cells co‐expressing CDK9‐mCherry and CRTC2‐eGFP or R>A‐eGFP and treated with DMSO or FSK (10 µ m ) for 1 h (left). The dotted line area indicates the nucleus. Quantification of cells with CDK9‐mCherry foci upon treatment with DMSO or FSK (right). b) Immunofluorescence analysis of co‐condensates of CRTC2‐IDR‐mCherry‐CRY2 with CDK9 or CycT1 in 293T cells before or after blue light stimulation (left). Quantification of cells with CRTC2‐IDR co‐condensates with CDK9 or CycT1 (right). c) Live‐cell imaging of 293T cells co‐expressing CycT1‐CFP, HEXIM1‐RFP, CRTC2‐eGFP, and R>A‐eGFP as indicated. d) Quantification of cells containing CycT1‐CFP foci. e) RNA‐IP assay examining the association between FLAG‐CDK9 and 7SK snRNA in 293T cells co‐expressing HA‐CRTC2 or HA‐R>A and treated with DMSO or FSK. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Live Cell Imaging, Expressing, Immunofluorescence

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 forms nuclear droplets in cystic epithelial cells. a) Western blot analysis of CRTC2 levels in kidneys from WT and Pkd1 −/− mice ( n = 3). b) Representative immunohistochemistry (IHC) images of CRTC2 in kidneys of WT and Pkd1 −/‐ mice (left). Quantification of the signal intensity of nuclear CRTC2 (right) ( n = 5). Cy, large cyst. c) Representative IF images of CRTC2 in the kidney of WT and Pkd1 −/‐ mice. Cy, large cyst. d) Representative IHC images of CRTC2 in kidneys from healthy people ( n = 5) and ADPKD patients ( n = 19). e) Quantification of the signal density of nuclear CRTC2 of (d). f) Correlation between the signal intensity of nuclear CRTC2 and eGFR in patients with ADPKD. Pearson's correlation coefficients are displayed for each graph. p values for (f) were determined using linear regression analysis. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. *** p < 0.001, * p < 0.05. Scale bar, 20 µm (b–d).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Western Blot, Immunohistochemistry

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: Loss of CRTC2 delays cyst formation in an ADPKD mouse model. a) Experimental design of the ADPKD mouse model. b) Representative kidneys from mice at P29 with indicated genotype. ( n = 8 or 9 biologically independent mice per group). c) KW/BW ratios of the indicated groups of mice in the ADPKD model. The number below each bar refers to the number of samples analyzed. d) Hematoxylin and eosin (H&E) staining of kidney sections from mice at P29 with indicated genotype. ( n = 8 or 9 biologically independent mice per group). e) Cystic index of H&E‐stained kidneys from mice at P29 with indicated genotype. The number below each bar refers to the number of samples analyzed. f) Plasma BUN levels of mice from the indicated groups. The number below each bar refers to the number of samples analyzed. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001. Scale bar, 2 mm (b,d).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Staining, Clinical Proteomics

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: Genome‐wide CRTC2 localization in human ADPKD cells. a) Representative immunofluorescence images of endogenous CRTC2 in WT 9–12 cells. The result is from more than three independent experiments. b) Western blot analysis of whole‐cell lysates from WT 9–12 cells with indicated treatment. The result is from more than three independent experiments. c) Genomic distribution of CRTC2 in WT 9–12 cells. d) Heatmaps of normalized ChIP‐seq signals for CRTC2, H3K4me1, and H3K4me3, H3K27ac. The rows show 3 kb flanking the CRTC2 peak center. e) Boxplots of the normalized counts of H3K4me1, H3K4me3, and H3K27ac signals at CRTC2 binding + or − peaks. f) Boxplots of CRTC2, H3K4me1, H3K4me3, and H3K27ac reads in the indicated groups. Low: CRTC2 reads < 39; medium: CRTC2 reads 40–70; high: CRTC2 reads > 71. g) ChIP‐seq tracks of CRTC2, H3K4me3, H3K27ac, and H3K4me1 on representative genes. h) Motif analysis of CRTC2 peaks. i) GO and KEGG pathway enrichment analyses of CRTC2‐binding genes. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. **** p < 0.0001, ** p < 0.01. Scale bar, 20 µm (a).

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Genome Wide, Immunofluorescence, Western Blot, ChIP-sequencing, Binding Assay

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 regulates the expression of cystogenesis‐associated genes in human ADPKD cells. a) Heatmap of gene expression in WT 9–12 cells transfected with sgControl or sgCRTC2. Rows show the Z scores calculated for each group. b) Volcano plots showing differentially expressed genes. c) Gene set enrichment analysis displaying CRTC2‐binding gene set enriched for genes downregulated in sgCRTC2 cells. NES, normalized enrichment score. d) Venn diagram showing the overlap of sgCRTC2 downregulated genes and CRTC2‐binding genes, referred to as CRTC2‐target genes. e) GO and KEGG pathway enrichment analyses of CRTC2‐target genes. f) Heatmap of expression values of genes enriched in the indicated processes. Rows show the Z scores calculated for each group. g) RT‐qPCR analysis of mRNA levels of representative CRTC2‐target genes in kidneys from the indicated mouse groups. Data are presented as means ± SEM. The unpaired Student's two‐sided t ‐test was used for statistical analysis. **** p < 0.0001. These results are from three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Expressing, Gene Expression, Transfection, Binding Assay, Quantitative RT-PCR

Journal: Advanced Science

Article Title: cAMP‐Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

doi: 10.1002/advs.202104578

Figure Lengend Snippet: CRTC2 activates cystogenesis‐associated genes by promoting paused Pol II release. a) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in the kidneys of WT and Pkd1 −/‐ mice ( n = 3). b) Immunofluorescence analysis of CRTC2 and CycT1 co‐localization in WT 9–12 cells (left). Line scan of the dashed line in the merged inset image (right). c) Western blot analysis of CRTC2 expression in WT 9–12 cells transfected with the indicated sgRNAs and constructs. d) Immunofluorescence analysis of ectopically expressed TY1‐CRTC2 and TY1‐CRTC2‐R>A in WT 9–12 cells with endogenous CRTC2 knocked out. e) RT‐qPCR analysis of representative CRTC2‐target genes in WT 9–12 cells transfected with the indicated sgRNAs and constructs. f) Cumulative curve of Pol II pausing index (PI) for CRTC2 target genes in normal and ADPKD kidney tissues. g) ChIP‐qPCR analysis of CycT1, Pol II Ser2P, and Pol II occupancy on LDHA and PLAU genes in WT 9–12 cells with endogenous CRTC2 replaced by TY1‐CRTC2 or TY1‐CRTC2‐R>A mutant. The inset bar graphs indicate the Pol II pausing index for LDHA and PLAU genes, respectively. h) Schematic illustration of how CRTC2 triggers the productive elongation of cystogenesis‐associated genes, ultimately leading to ADPKD progression. Data are presented as means ± SEM. The unpaired two‐sided Student's t ‐test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar, 20 µm (a,b). All results are from more than three independent experiments.

Article Snippet: Protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes and immunoblotted with primary antibodies against CRTC2 (Proteintech, 12497‐1‐AP, dilution 1:1000), CDK9 (Santa Cruz, sc‐13130, dilution 1:1000), HEXIM1 (Proteintech, 15676‐1‐AP, dilution 1:5000), HA (Cell Signaling Technology, 3724, dilution 1:1000), FLAG (Sigma, A8592, dilution 1:5000), Myc (Cell Signaling Technology, 2278, dilution 1:2000), α ‐tubulin (Proteintech, 11224‐1‐AP, dilution 1:5000), and TY1 (Invitrogen, MA5‐23513, dilution 1:2000).

Techniques: Immunofluorescence, Western Blot, Expressing, Transfection, Construct, Quantitative RT-PCR, ChIP-qPCR, Mutagenesis

Journal: Cell reports

Article Title: Identification of molecular signatures defines the differential proteostasis response in induced spinal and cranial motor neurons

doi: 10.1016/j.celrep.2024.113885

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RNAse-Free DNase , Qiagen , 79254.

Techniques: Recombinant, Knock-Out, Blocking Assay, DC Protein Assay, Transcriptome Wide Gene Expression, Control, Software, Suspension, Protein Extraction, Mass Spectrometry, Fluorescence, Membrane, Electrophoresis, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Synaptic Activity Drives a Genomic Program That Promotes a Neuronal Warburg Effect *

doi: 10.1074/jbc.M116.761106

Figure Lengend Snippet: Genome-wide transcriptome analysis reveals nuclear encoded mitochondrial genes that are regulated by synaptic activity Transcriptome data from a previous study from our group were compared with the MitoCarta database of mitochondrially localized proteins. The table lists genes with evidence for mitochondrial localization whose expression is changed more than 1.5-fold by bicuculline-induced AP bursting. For each gene, the official symbol and full name as well as fold change compared with control after 2, 4, and 6 h of treatment with 50 μ m bicuculline and after 2 and 4 h of bath application of 20 μ m glutamate are listed. Additional activity-regulated genes with a role in energy metabolism were identified from transcriptome analyses. Transcriptome analyses were performed with Affymetrix GeneChips (4; n = 3) and RNA-Seq ( n = 2). NA, not applicable.

Article Snippet: To determine changes in mRNA expression, total RNA was extracted from primary cultures and hippocampal tissue using an RNeasy mini kit (Qiagen) with additional on-column DNase I digestion. cDNA was synthesized from 1 μg of total RNA using a high capacity cDNA reverse transcription kit (Applied Biosystems). qRT-PCR was done on an ABI7300 thermal cycler using universal qRT-PCR master mix (Applied Biosystems) with the following TaqMan gene expression assays (Applied Biosystems): 18s rRNA (Mm03928990_g1), Abcb10 (Mm00497926_m1), Abhd10 (Mm00552934_m1), Acsl4 (Mm00490331_m1), Actb (Mm00607939_s1), Aqp4 (Rn00563196_m1), Atf3 (Rn00563784_m1), Atpaf1 (Mm00619286_g1, Rn00619284_m1), Dguok (Mm00443332_m1), Gapdh (Mm99999915_g1), Gfap (Mm01253031_m1), Gusb (Mm00446953_m1, Rn00566655_m1), Mrpl19 (Mm00452754_m1), Mrpl44 (Mm00452819_m1), Pdk3 (Mm00455220_m1), Slc2a1 (Glut1, Mm00441480_m1), Slc2a3 (Glut3, Mm00441483_m1), Slc16a1 (Mct1, Mm01306379_m1, Rn00562332_m1), Slc16a7 (Mct2, Rn00568872_m1), Sod1 (Mm01344233_g1), Tfam (Mm00447485_m1), Tfb2m (Mm01620397_s1), and Ucp1 (Mm01244861_m1).

Techniques: Genome Wide, Activity Assay, Expressing, Control, RNA Sequencing, RNA Binding Assay

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) CRITR-seq vector with a type III interferon reporter. Functional gRNA is constitutively transcribed from the U6 promoter. gRNA sequence is also present in the reporter mRNA transcribed from the IFNL1 promoter, serving as a barcode indicating which edit occured in the transcribing cell. SIN LTR = self-inactivating long terminal repeat, LNGFR = low-affinity nerve growth factor receptor, WPRE = woodchuck hepatitis virus posttranscriptional regulatory element. (B) Model of amplicon sequencing and data analysis of CRITR-seq screen. Amplicon sequencing is performed on the gRNA-containing regions of the genomic DNA and polyadenylated mRNA. The mRNA/gDNA ratio for each gRNA sequence represents the normalized IFNL1 transcription levels for that guide. Graph represents a distribution of all gRNAs based on their mRNA/gDNA ratio, with individual examples highlighted as colored points plotted based on their hypothetical Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) score. (C) Workflow for the CRITR-seq screen in this study. 3 libraries were generated independently, starting from PCR-amplifying the gRNAs out of the GeCKO library . A clonal line of Cas9-expressing A549 cells was transduced with lentivirus carrying the CRITR-seq vector at an MOI of 1.5, with an assumption that most gRNAs would not affect the interferon induction phenotype, so very little epsitasis would complicate our measurements. After allotting 10 days for gene editing, edited cells were infected with NS1 mut influenza A virus at an MOI of 2 based on qPCR titer.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Plasmid Preparation, Functional Assay, Sequencing, Virus, Amplification, Genome Wide, CRISPR, Knock-Out, Generated, Expressing, Transduction, Infection

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) Three CRITR-seq libraries were generated by cloning the GeCKO library gRNA sequences into the CRITR-seq vector three independent times. Amplicon sequencing was performed on the region containing the gRNA in the plasmid library, the genomic DNA of the transduced A549 cells, and the mRNA from the transduced cells after 8 hour infection by NS1 mut . gRNAs were identified for reads matching the CRITR-seq vector for the 10 nucleotides upstream and 13 nucleotides downstream of the gRNA sequence, requiring perfect matching. (B-D) Distribution of gRNAs by count in the plasmid (B), genomic DNA (C), and RNA (D) libraries.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Generated, Cloning, Plasmid Preparation, Amplification, Sequencing, Infection

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) Distribution of gRNAs across all 3 replicates of the CRITR-seq screen, based on mRNA/gDNA ratio. gRNAs with less than 25 reads in genomic DNA in any replicate were excluded from analysis. Read counts across replicates were normalized based on the non-targeting gRNAs from each replicate. (B) Distribution of genes based on the median mRNA/gDNA ratio for surviving guides targeting that gene across all 3 replicates. Individual genes from the RIG-I signaling pathway, shown in (C), are plotted based on their MAGeCK robust ranking aggregation score for depletion in the mRNA. MAGeCK statistical scores were calculated using non-targeting gRNAs as the null distribution, with depletion in the mRNA as the alternative hypothesis. (C) RIG-I signaling pathway. Genes/proteins in blue are considered essential for interferon induction through this pathway, while genes/proteins in green may be partially redundant. IRF7 was excluded from this analysis because in epithelial cells it is expressed at very low levels prior to interferon signaling and likely does not contribute to interferon transcription at this early time point , . (D) mRNA/gDNA ratios from (B), subsetted by gene category. “Proviral” genes were identified by Li et al. in a CRISPR screen for genes required for influenza infection in A549 cells. “Early” genes are a subset of the proviral genes annotated to be involved in viral entry, nuclear import, viral transcription/replication, or nuclear export. Proviral and Early genes tested are listed in Table S1. RIG-I pathway genes are those shown in (C). Solid lines represent the median; dotted lines represent the first and third quartiles. * indicates ANOVA p<0.05, post-hoc Tukey’s test q<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: CRISPR, Infection

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) Same distribution of genes from CRITR-seq screen as in , but with the top 3-ranked genes enriched in the mRNA highlighted. MAGeCK statistical scores here were calculated with enrichment in the mRNA as the alternative hypothesis, using non-targeting gRNAs as null distribution. (B) A549 cells were transfected with Cas9-gRNA ribonucleoprotein complexes with gRNAs targeting the indicated genes. After passaging 10 days to allow for gene editing, cells were infected with NS1 mut at a genome-corrected MOI of 2. RNA was harvested at 8 hours post-infection for qPCR analysis of IFNB1 and IFNL1 transcripts. NTC = non-targeting control. (C-D) A549 cells were treated with siRNA for 9 days and then infected with NS1 mut at a genome-corrected MOI of 1 (C) or WT WSN at an infectious MOI of 1 (D). RNA was harvested 8 hours post-infection for qPCR analysis. The same results were obtained for the WSN NS1 stop virus (with WSN genetic background for all segments), shown in . Validation of NELFB knockdown is shown in . (E) A549 cells were treated with siRNA for 9 days before harvesting RNA for qPCR. n.d. = not detected. Validation of NELFB knockdown is shown in . (F) A549 cells were treated with siRNA for 9 days and transfected with 50 ng poly(I:C) for 8 hours before harvesting RNA for qPCR. Validation of NELFB knockdown is shown in . Biological replicates are shown as individual data points, with lines representing the means. One-tailed t-test (B) with increased expression as the alternative hypothesis, or two-tailed t-tests (C-F) were performed to compare each treatment with the non-targeting control. n=3 (B,D-F) or n=4 (C). * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Transfection, Passaging, Infection, Control, Virus, Biomarker Discovery, Knockdown, One-tailed Test, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A) RNA from experiment performed in and , with HA transcripts measured by qPCR. The same results were obtained for the WSN NS1 stop virus, shown in . NTC = non-targeting control. (B) A549 cells were treated with siRNA for 9 days. Cells were infected with PB1 455:350 at an infectious MOI of 1, with or without 100 nM baloxavir acid added at time of infection. RNA was harvested 8 hours post infection for analysis by qPCR. Validation of NELFB knockdown is shown in . (C) An A549 IFNL1 reporter cell line was treated with siRNA for 9 days and infected with NS1 mut at a genome-corrected MOI of 1. 13 hours post infection, cells were stained for the viral protein M2 and fixed for flow cytometry. Graph shows distribution of M2 staining normalized for unit area, for one representative replicate. Full flow data shown in . (D) A549 cells were treated with either of 2 different NELFB -targeting siRNAs, a non-targeting control, or no siRNA, with 2 biological replicates per treatment. After 9 days of siRNA treatment, cells were infected with NS1 mut at a genome-corrected MOI of 2. RNA was harvested 8 hours post infection, and 5’ RACE was performed on polyadenylated mRNAs, followed by sequencing. Cap-snatched sequence length refers to the number of nucleotides between the template switch oligo sequence and the +1 position of the flu mRNA sequence. Violin plots contain box plots for each sample, and the median of all samples is represented by the gray dotted line. Violin plot whiskers extend to the most extreme points in the dataset, excluding the top 2% of lengths. Validation of NELFB knockdown phenotype is shown in . (E) A549 cells were treated with siRNA for 4 days and infected with PB1 455:350 at an infectious MOI of 1. RNA was harvested 8 hours post infection for analysis by qPCR. (F) A549 cells were treated with siRNA 9 days before infection with WT WSN at an infectous MOI of 5. Media was replaced with fresh IGM 2 hours post infection. 14 hours post infection, viral supernatant was collected and cells were lysed for RNA extraction. Reverse transcription was performed using universal influenza primers for the RNA from the supernatant, and random hexamer primers for the RNA from the cell lysate. Further qPCR analysis is shown in . For panels A-B and E-F, biological replicates are shown as individual data points, with lines representing the means. Two-tailed t-tests were performed to compare targeted siRNA with the non-targeting control. n=4 (A, left) or n=3 (A, right; B; E-F). Benjamini-Hochberg multiple hypothesis correction was performed for panels B and F. * indicates p<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Virus, Control, Infection, Biomarker Discovery, Knockdown, Staining, Flow Cytometry, Sequencing, RNA Extraction, Reverse Transcription, Random Hexamer, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: A549 IFNL1 reporter cells were treated for 9 days with siRNA and infected, or not, with NS1 mut at a genome-corrected MOI of 1. 13 hours post infection, cells were stained for the viral protein M2 and fixed for flow cytometry. (A) Individual replicates shown. The threshold for IFNL1 + or M2 + cells was set at the fluorescence level for which an average of 0.1% of uninfected cells would be called as positive events. Interferon-positive events colored in orange. For visualization, data was subsetted to 5000 events to show equivalent numbers between conditions. (B) Mean fluorescence intensity (MFI) of M2 staining. Two-tailed t-test was performed to compare targeted siRNA with the non-targeting control, n=3, * indicates p<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Infection, Staining, Flow Cytometry, Fluorescence, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: A549 cells were treated with either of 2 different NELFB -targeting siRNAs, a non-targeting control, or no siRNA, with 2 biological replicates per treatment. After 9 days of siRNA treatment, cells were infected with NS1 mut at a genome-corrected MOI of 2. RNA was harvested 8 hours post infection for 5’ RACE and sequencing and qPCR analysis. qPCR results shown here confirm NELFB knockdown and a consistent phenotype of increased viral RNA and interferon production in the samples treated with NELFB siRNA. Biological replicates are shown as individual data points, with lines representing the means. Two-tailed t-tests were performed to compare each sample with the untreated condition and with the non-targeting control, n=2. * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Control, Infection, Sequencing, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A-B) Same experiment as in , with different RNAs measured by qPCR. A549 cells were treated with siRNA for 9 days before infection with WT WSN at an infectious MOI of 5. Media was replaced with fresh IGM 2 hours post infection. 14 hours post infection, viral supernatant was collected and cells were lysed for RNA extraction of both. Reverse transcription was performed using universal influenza primers for the RNA from the supernatant, and random hexamer primers for the RNA from the cell lysate. Biological replicates are shown as individual data points, with lines representing the means. Two-tailed t-tests were performed to compare targetign siRNA with the non-targeting control, n=3. Benjamini-Hochberg multiple hypothesis correction was performed for panel A. * indicates p<0.05.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Infection, RNA Extraction, Reverse Transcription, Random Hexamer, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A-C) Wild-type A549 cells (A-B) or a RIG-I -knockout cell line derived from a single cell clone (C) were treated with siRNA for 9 days. Cells were infected with NS1 mut at a genome-corrected MOI of 1, with or without 100 nM baloxavir acid added at time of infection. RNA was harvested 8 hours post infection for qPCR analysis. NTC = non-targeting control. We note the RIG-I -knockout cell line has some residual RIG-I expression observed by Western blot after flu infection . HA qPCR measurements in these cells are shown in . (D) Flow cytometry experiment from , with zsGreen IFNL1 reporter results shown. Left: % IFNL1 + cells determined as the percent of cells positive for zsGreen expression. Right: mean fluorescence intensity (MFI) of zsGreen for IFNL1 + cells. Full flow data shown in . (E) Cells from (D) were divided into bins based on levels of M2 staining. % IFNL1 + cells plotted for each bin. Bins with less than 100 events were removed from plotting and analysis. Points represent biological replicates, with lines indicating the means. Two-tailed t-tests were performed comparing the NELFB siRNA samples with the non-targeting control for each treatment condition (A-D) or for each M2 expression bin (E), n=3. * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Knock-Out, Derivative Assay, Infection, Control, Expressing, Western Blot, Flow Cytometry, Fluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: We transfected A549 cells with Cas9 ribonucleoprotein using a gRNA targeting RIG-I . After performing dilution cloning to isolate single cells, the clonal-derived line was infected with NS1 mut at a genome-corrected MOI of 2 for 24 hours. Infected and uninfected A549 or A549 RIG-I knockout cells were lysed for SDS-PAGE and Western blot.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Transfection, Cloning, Derivative Assay, Infection, Knock-Out, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

doi: 10.1101/2024.11.14.623683

Figure Lengend Snippet: (A-B) A549 cells were infected, or not, with PB1 455:350 , at an infectious MOI of 1, with or without 100 nM baloxavir acid or 100 nM pimodivir added 2 hours prior to infection. RNA was harvested 14 hours post infection for qPCR analysis. (C-D) A549 cells were infected with a high-defective population of WT WSN at an MOI of 2 based on qPCR titer. For drug treatments, cells were treated with 10 nM baloxavir acid and/or 50 μg/mL cycloheximide (CHX) at time of infection. RNA was harvested 9 hours post infection for qPCR analysis. Points represent biological replicates, with lines indicating the means. For A and B, two-tailed t-tests were performed comparing each treatment with the uninfected sample and with the infected sample without inhibitors, n=3. For C and D, two-tailed t-tests were performed comparing presence and absence of baloxavir, for each CHX condition, n=3. * indicates p<0.05 after Benjamini-Hochberg multiple hypothesis correction.

Article Snippet: The following cell lines were used in this study: HEK293T (human embryonic kidney, female; ATCC CRL-3216), MDCK-SIAT1 (variant of the Madin Darby canine kidney cell line overexpressing SIAT1, female cocker spaniel; Sigma-Aldrich 05071502), and A549 (human lung epithelial carcinoma, male; ATCC CCL-185).

Techniques: Infection, Two Tailed Test