Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 1 Elk4 is expressed and downregulated in activated mast cells. (A) Scatterplot displaying the induced (red) and downregulated genes (green) after FcϵRI-mediated stimulation in human mast cells (|Log2 fold change|≥1, adj p<0.05). The transcripts per kilobase of exon model per million mapped reads (TPM) were extracted from the GSE107316 dataset. (B) Gene ontology analysis of FcϵRI crosslinking-induced and FcϵRI crosslinking- downregulated genes by using the MSigDB hallmark gene set signatures and TCF target gene signature. Hypergeometrical tests were used to assess statistical significance. Bonferroni-adjusted p values are shown. (C) The mRNA levels of ELK1, ELK3, and ELK4 in unstimulated, IgE-sensitized, and IgE-DNP/HSA-stimulated human mast cells are shown. TPM data were extracted from the GSE107316 dataset. (D) qPCR analysis of Elk1, Elk3, and Elk4 mRNA expression in unstimulated, IgE-DNP/HSA-stimulated and Compound 48/80-stimulated BMMCs. One-way ANOVA with Dunnett’s multiple comparison test was used to assess statistical significance. Bar, mean; error bar, SD; n = 3; *p<0.05; **p<0.01. (E) Western blot analysis of ELK4 protein expression in unstimulated, IgE-DNP/HSA-stimulated and Compound 48/80-stimulated BMMCs.

Article Snippet: The following antibodies were used for endogenous co-IP and WB analysis: SIRT6 (Cell Signaling Technology, 12486), ELK4 Frontiers in Immunology 03 (Atlas antibodies, HPA028863), and MITF (Cell Signaling Technology, 97800).

Techniques: Expressing, Comparison, Western Blot

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 2 Elk4 deficiency leads to cell cycle arrest in BMMCs. (A) The proliferation of WT BMMCs and Elk4 KO BMMCs was assessed by CCK8 assay at day 1, day 3 and day 5. Two-way ANOVA with Dunnett’s multiple comparison test was used to assess statistical significance. Bar, mean; error bar, SD; n=3; *p, < 0.05; ***, p<0.001. (B, C) Cell cycle analysis of WT BMMCs and Elk4 KO BMMCs cultured in the presence of IL3 with or without SCF. Flow cytometry data (B) and statistical data (C) are shown. Bar, mean; error bar, SD; n=3; *p, < 0.05; **p<0.01. (D) Percentage of PCMCs in CD45-positive cells from the peritoneal cavities of WT and Elk4 KO mice. Cells were stained with CD45-eFluor 450, CD117-FITC, and FcϵRI-PECY7 and analyzed by flow cytometry. (E) Toluidine blue staining of skin tissues from WT and Elk4 KO mice. The mast cell number (blue)/high-power field (HPF) was used for statistical analysis. Bar, mean; error bar, SD; n=4; ***p<0.001.

Article Snippet: The following antibodies were used for endogenous co-IP and WB analysis: SIRT6 (Cell Signaling Technology, 12486), ELK4 Frontiers in Immunology 03 (Atlas antibodies, HPA028863), and MITF (Cell Signaling Technology, 97800).

Techniques: CCK-8 Assay, Comparison, Cell Cycle Assay, Cell Culture, Flow Cytometry, Staining, Cytometry

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 3 Hdc, Tnfa, Il6, Ccl3, and Ccl4 mRNA expression was impaired in Elk4 KO BMMCs in response to FcϵRI-mediated and Compound 48/80-induced activation. (A) qPCR analysis of Hdc, Il6, Tnfa, Ccl3, and Ccl4 mRNA levels in BMMCs derived from Elk4 wild-type (WT), heterozygous (HZ), and homozygous (KO) mice in response to FcϵRI-mediated activation. BMMCs were sensitized with anti-DNP-IgE(0.5 mg/ml) overnight and stimulated with DNP-HSA (100 ng/ml) for 1 hour. Bar, mean; error bar, SD; n=3; ****p<0.0001. (B) qPCR analysis of the Hdc, Ccl3, and Ccl4 mRNA levels in BMMCs derived from Elk4 WT, HZ, and KO mice in response to Compound 48/80-induced activation. One-way ANOVA with Dunnett’s multiple comparison test was used to assess statistical significance in this figure. BMMCs were stimulated with Compound 48/80 (5 mg/ml) for 1 hour. Bar, mean; error bar, SD; n=3; *p < 0.05; **p<0.01; ***p<0.001.

Article Snippet: The following antibodies were used for endogenous co-IP and WB analysis: SIRT6 (Cell Signaling Technology, 12486), ELK4 Frontiers in Immunology 03 (Atlas antibodies, HPA028863), and MITF (Cell Signaling Technology, 97800).

Techniques: Expressing, Activation Assay, Derivative Assay, Comparison

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 4 Elk4 deficiency promotes degranulation and histamine release. (A) Time course and dose-dependent curve detection of the beta-hexosaminidase releases. BMMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight, then stimulated with DNP-HSA (10, 100, 1000 ng/ml) for 60min (Left) or stimulated with DNP-HSA (100 ng/ml) for 5min, 15min, 30min and 60min (Right). Bar, mean; error bar, SD; n=4; **p<0.01. (B) The total contents of beta-hexosaminidase in Elk4 WT, HZ and KO BMMCs are shown. Bar, mean; error bar, SD; n=3; *p < 0.05; **p<0.01. (C, D) Degranulation of Elk4 WT, HZ and KO PCMCs was assessed by beta-hexosaminidase release assay. PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight and stimulated with DNP-HSA (100 ng/ml) for 1 hour. The released contents (A) and the total contents (B) of beta-hexosaminidase in Elk4 WT, HZ and KO PCMCs are shown. Bar, mean; error bar, SD; n=2; **p<0.01. (E, F) Degranulation of Elk4 WT, HZ and KO BMMCs (E) and PCMCs (F) was assessed by cell-surface LAMP1 staining using flow cytometry. Both BMMCs and PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight and stimulated with DNP-HSA (100 ng/ml) for 1 hour before LAMP1 staining. Bar, mean; error bar, SD; n=3; *p<0.05.

Article Snippet: The following antibodies were used for endogenous co-IP and WB analysis: SIRT6 (Cell Signaling Technology, 12486), ELK4 Frontiers in Immunology 03 (Atlas antibodies, HPA028863), and MITF (Cell Signaling Technology, 97800).

Techniques: Release Assay, Staining, Cytometry

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 5 The anaphylactic response in mice was alleviated in Elk4-deficient mice. (A) Images showing the ear pinnae of Elk4 WT and KO mice. Evans blue dye extravasation from the ears of Elk4 WT and KO mice was examined 30 min after intravenous DNP-HSA (containing 1% Evans blue) administration. Evans blue dye extravasation was quantified by measuring the optical density at 610 nm (OD610nm)/weight. Bar, mean; error bar, SD; n=6; ***p<0.001. (B, C) WT and Elk4 KO mice were sensitized with 10 mg anti-DNP-IgE and challenged with 100 mg DNP-HSA. Changes in body temperature and serum levels of IL-6, TNFa and histamine were determined. Bar, mean; error bar, SD; n=3-5; *p<0.05; ***p<0.001. (D) HE staining, IHC analysis of MUC5AC and toluidine blue staining of lung sections from Elk4 WT and KO mice with OVA-induced asthma. The area of the bronchiole wall (Wa) and the perimeter of the bronchiole basement membrane were analyzed by ImageJ. Student’s t test was used to assess statistical significance in the figure. Bar, mean; error bar, SD; n=3; **p<0.01; ***p<0.001.

Article Snippet: The following antibodies were used for endogenous co-IP and WB analysis: SIRT6 (Cell Signaling Technology, 12486), ELK4 Frontiers in Immunology 03 (Atlas antibodies, HPA028863), and MITF (Cell Signaling Technology, 97800).

Techniques: Staining, Membrane

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 6 Elk4 deficiency affects the FcϵRI-mediated transcriptional response in BMMCs. (A) Scatterplot displaying the induced (red) and downregulated genes (green) before and after FcϵRI-mediated stimulation in WT BMMCs (|Log2 fold change|≥1, adj p<0.05). (B) Gene ontology analysis of FcϵRI crosslinking-induced and FcϵRI crosslinking-downregulated genes in WT BMMCs by using the MSigDB hallmark gene set signatures and TCF target gene signature. Hypergeometrical tests were used to assess statistical significance. Bonferroni-adjusted p values are shown. (C) Heatmap representation of the relative mean expression levels of the 431 IgE-DNP/HSA-induced genes in Elk4 WT and KO BMMCs. (D) Identification of ELK4- dependent genes by comparison with the IgE-DNP/HSA induction ratio in Elk4 WT and KO BMMCs. ELK4-dependent genes (red, purple) exhibited a systematic relationship between their degree of induction in the two contexts, whereas the others (grey) did not. Slope and Spearman r value are indicated. (E) Gene ontology analysis of the ELK4-dependent (fold change>1.5 or <0.66, adj p<0.05 in Elk4 KO BMMCs) FcϵRI-crosslinking induced genes in BMMCs by using the MSigDB hallmark gene set signatures. Hypergeometrical tests were used to assess statistical significance. Bonferroni- adjusted p values are shown. (F) Heatmap representation of the relative mean expression levels of the differentially expressed genes between unstimulated Elk4 WT and KO BMMCs. Gene ontology analysis of the differentially expressed genes was further performed by using the MSigDB hallmark gene set signatures and MITF and TCF target gene signatures. Hypergeometrical tests were used to assess statistical significance. Bonferroni-adjusted p values are shown.

Article Snippet: The following antibodies were used for endogenous co-IP and WB analysis: SIRT6 (Cell Signaling Technology, 12486), ELK4 Frontiers in Immunology 03 (Atlas antibodies, HPA028863), and MITF (Cell Signaling Technology, 97800).

Techniques: Expressing, Comparison

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 7 ELK4 interacts and might cooperate with MITF and SIRT6 to regulate mast cell activation and degranulation. (A) Coimmunoprecipitation analysis of endogenous MITF and ELK4 proteins in MC/9 mast cells. (B) qPCR analysis of the Hdc, Tnfa, Ccl3, and Ccl4 mRNA levels in BMMCs treated with ML329 in response to FcϵRI-mediated stimulation. One-way ANOVA with Dunnett’s multiple comparison test was used to assess statistical significance. Bar, mean; error bar, SD; n=3; *p < 0.05; ****p<0.0001. (C) Coimmunoprecipitation analysis of endogenous SIRT6 and ELK4 proteins in MC/9 mast cells. (D) qPCR analysis of Syngr1, Exoc3l1, Cadm1, and Vcl mRNA levels in Elk4-deficient BMMCs. Student’s t test was used to assess statistical significance. Bar, mean; error bar, SD; n=3; **p<0.01. (E) qPCR analysis of Syngr1, Exoc3l1, Cadm1, and Vcl mRNA levels in BMMCs treated with OSS-128167. * indicates p<0.05; ** indicates p <0.01;***indicates p<0.001. One-way ANOVA with Dunnett’s multiple comparison test was used to assess statistical significance. Bar, mean; error bar, SD; n=3; *p < 0.05; **p<0.01; ****p<0.0001. (F) Schematic diagram depicting the downregulation of Elk4 expression in activated mast cells and the effect and potential mechanism of ELK4 on mast cell activation.

Article Snippet: The following antibodies were used for endogenous co-IP and WB analysis: SIRT6 (Cell Signaling Technology, 12486), ELK4 Frontiers in Immunology 03 (Atlas antibodies, HPA028863), and MITF (Cell Signaling Technology, 97800).

Techniques: Activation Assay, Comparison, Expressing

Journal: Nature Communications

Article Title: Reprogramming of cis -regulatory networks during skeletal muscle atrophy in male mice

doi: 10.1038/s41467-023-42313-3

Figure Lengend Snippet: a Heatmap (Z-score) illustrates average chromVAR motif activity for each cell type in both normal and denervated muscles. Refer to Supplementary data for atrophy-related motifs and their best-matched TFs specific to each cell type. b Dot plots display the differential motif activities (left) and corresponding TF expression (right) in normal and denervated myonuclei. Significance was evaluated with a Wilcoxon Rank Sum two-side test, n = 3 mice per group containing 14,388 nuclei in a joint analysis. c Heatmap presents select myonuclei-specific regulon activities in normal and denervated muscles, as identified by intersecting ChromVar and SCENIC results. The comprehensive list of TFs (regulons) is available in Supplementary data . The SCENIC algorithm binarized regulon activity as “On” (black) or “Off” (white). d UMAP plots showcase the upregulation of Elk4 and its target genes (upper), or the downregulation of Ar and its target genes (lower) in response to denervation. e H3K27ac ChIP-seq profile of normal and denervated muscles. Left: the read count frequency in selected range around TSS. Right: heatmaps of normalized H3K27ac tag densities at differentially H3K27-acetylated regions. f Heatmap displays candidate enhancers with either increased (left) or decreased (right) H3K27ac signals in denervated muscles. For a complete list of inferred candidate enhancers, refer to Supplementary data and . g , h Left: Fragment coverage tracks showing chromatin accessibility, gene expression, and H3K27ac ChIP-seq signals. Red arrowhead indicates the tested regions of enhancers. Right: Representative image of in vivo enhancer activity visualized by X-Gal staining in tibialis anterior (TA) muscles from 3 normal and 3 denervated mice. Scale bar: 50 μm. Source data are provided as a Source data file.

Article Snippet: The untagged or green-fluorescent-protein (GFP) tagged mouse Elk4 (NM_001376954.1) pAAV expression vectors (VB900124-9241zuq, VB900138-4352whw), GFP marked Elk4 pAAV-shRNA knockdown vectors (VB900138-4353mwt, VB900129-1910enq, VB900138-4354tjw), and related control vectors (VB010000-9394npt, VB010000-0023jze) were purchased form VectorBuilder (Chicago, IL, USA).

Techniques: Activity Assay, Muscles, Expressing, ChIP-sequencing, Gene Expression, In Vivo, Staining

Journal: Nature Communications

Article Title: Reprogramming of cis -regulatory networks during skeletal muscle atrophy in male mice

doi: 10.1038/s41467-023-42313-3

Figure Lengend Snippet: a Upper: Violin plot of Elk4 expression across myonuclei types. Lower: Tracks show chromatin accessibility, Elk4 expression, and H3K27ac profile at the Elk4 locus. b Line plots track Elk4 activity dynamics from normal to denervation. The solid line represents the Local Polynomial Regression fit, with the shaded region indicating the 95% confidence interval. c Quantitative RT-PCR analysis of Elk4 mRNA in normal and denervated GAS muscle ( n = 6). d Immunofluorescence staining of Elk4 in normal and denervated GAS muscle. Scale bar: 25 μm. e TF footprinting plot demonstrating Elk4 motif activity in normal and denervated myonuclei. f Transcriptional network map of Elk4 regulons (left) and line plots of gene activity dynamics for Elk4 target genes across pseudotime. g Volcano plot of transcriptomic changes in C2C12 cells post Elk4 overexpression. Red and blue dots signify up- and down-regulated genes with log2-fold change thresholds of ±1 and p -value < 0.05, analyzed using a two-sided Fisher’s exact test in EdgeR v3.12.1. h Fragment coverage plot showing Elk4 motif within the promoter region (2000bp from TSS) of the Tgfb1 locus. Predicted scores are shown in parentheses. i Immunoblotting and quantitative analysis of ELK4, TGFB1, and p-SMAD3 in C2C12 cells with or without Elk4 overexpression ( n = 6). j Immunoblotting and quantitation of ELK4, TGFB1, and p-SMAD3 in normal and denervated GAS muscle ( n = 8). k Fluorescence images displaying changes in C2C12 myotubes (transfected with AAV1-Scramble-GFP or Elk4 siRNA-GFP) in response to ionomycin (IONO, 1 mM, 48 h). Scale bar: 50 μm. l Immunoblotting (left) and quantitative analysis (right) of ELK4, TGFB1, and p-SMAD3 for experiments in ( k ) ( n = 4). m Quantitative RT-PCR analysis of atrophy-related genes in normal or ionomycin-treated C2C12 myotubes with or without Elk4 knockdown ( n = 4). All bar graphs present quantitative data as mean ± SEM. “ n ” denotes the number of biological replications ( i , l , m ) or mice/group ( c , j ). Significance was assessed using a two-side Wilcoxon rank-sum test ( c , i , j ) or two-side one-way ANOVA with post hoc Tamhane’s multiple comparisons test ( l , m ). Source data are provided as a Source data file.

Article Snippet: The untagged or green-fluorescent-protein (GFP) tagged mouse Elk4 (NM_001376954.1) pAAV expression vectors (VB900124-9241zuq, VB900138-4352whw), GFP marked Elk4 pAAV-shRNA knockdown vectors (VB900138-4353mwt, VB900129-1910enq, VB900138-4354tjw), and related control vectors (VB010000-9394npt, VB010000-0023jze) were purchased form VectorBuilder (Chicago, IL, USA).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Immunofluorescence, Staining, Footprinting, Over Expression, Western Blot, Quantitation Assay, Fluorescence, Transfection, Knockdown

Journal: Nature Communications

Article Title: Reprogramming of cis -regulatory networks during skeletal muscle atrophy in male mice

doi: 10.1038/s41467-023-42313-3

Figure Lengend Snippet: a Left: Fluorescence images of TA muscles transfected with pAAV-GFP plasmid containing either scramble (Scr) shRNA or a combination of three pAAV-GFP plasmids each with a specific Elk4-targeting shRNA. Middle: Scatter-bar plot comparing the weight of transfected TA muscles. Right: Scatter-bar plot of the weight of ipsilateral (non-transfected) GAS muscles 14 days post-denervation. Data are presented as mean ± SEM ( n = 12 mice/group). Significance was determined by two-sided one-way ANOVA with post hoc Tamhane’s multiple comparisons test. Scale bar: 2 mm. b Cross-sectional images corresponding to samples in ( a ). “CSA” indicates the cross-sectional area. Scale bar: 25 μm. c Distribution of myofiber sizes derived from the samples in ( b ). d Quantitative analysis of the cross-sectional area based on the samples from ( b ). Data are presented as mean ± SEM ( n = 5 mice/group, significance was determined by two-sided one-way ANOVA with post hoc Tamhane’s multiple comparisons test). e Western blot analysis (left) and its quantification (right) using samples from ( a ). Data are shown as mean ± SEM ( n = 8 mice/group, significance was determined by two-sided one-way ANOVA with post hoc Tamhane’s multiple comparisons test). f Quantitative RT-PCR analysis of atrophy-related genes in TA muscle, comparing normal versus denervated conditions with or without Elk4 knockdown. Data are presented as mean ± SEM ( n = 5 mice/group, significance was determined by two-side one-way ANOVA with post hoc Tamhane’s multiple comparisons test). Source data are provided as a Source data file.

Article Snippet: The untagged or green-fluorescent-protein (GFP) tagged mouse Elk4 (NM_001376954.1) pAAV expression vectors (VB900124-9241zuq, VB900138-4352whw), GFP marked Elk4 pAAV-shRNA knockdown vectors (VB900138-4353mwt, VB900129-1910enq, VB900138-4354tjw), and related control vectors (VB010000-9394npt, VB010000-0023jze) were purchased form VectorBuilder (Chicago, IL, USA).

Techniques: Fluorescence, Muscles, Transfection, Plasmid Preparation, shRNA, Derivative Assay, Western Blot, Quantitative RT-PCR, Knockdown