Journal: The Journal of Biological Chemistry

Article Title: An essential signaling function of cytoplasmic NELFB is independent of RNA polymerase II pausing

doi: 10.1016/j.jbc.2023.105259

Figure Lengend Snippet: NELFC binding-deficient mutants of NELFB are retained in the cytoplasm . A , Super-resolution 3D structured illumination microscopy-based immunofluorescence image of NELFB ( blue ) and DAPI ( yellow ) in WT cells. Representative images from three independent repeats were shown. The scale bar represents 20 μm. B , immunofluorescent staining of NELFB and DAPI in WT, MT183, MT194, and MT486 cells. Representative images from three independent repeats were shown. The scale bar represents 20 μm. C , Western blotting of different NELF subunits in ectopic WT and MT194 clones. Tubulin and H3 are used as markers for cytosol and nuclear fractions, respectively. Two independent clones for each cell line were used and the experiments were done with two independent repeats. Asterisks indicate elevated levels of cytoplasmic NELFB and NELFE in MT194 cell clones. D , Western blotting of NELF subunits in NELFC-AID cells, following treatment of PBS or 0.5 mM IAA for 6 h. GAPDH, ATP5B, and LAMIN B1 were used as markers for cytosol, membrane, and nuclear fractions, respectively. Representative result is shown from three independent biological repeats. Asterisks indicate elevated levels of cytoplasmic NELFB and NELFE, despite the reduction of cytoplasmic NELFC. AID, auxin-induced protein degradation; DAPI, 4′,6-diamidino-2-phenylindole; IAA, indole-3-acetic acid; NELF, negative elongation factor.

Article Snippet: C-terminal mini-AID–tagged mouse NELFB and human NELFC were cloned into pLenti-6.3/V5-DEST-GFP (Addgene: 40125).

Techniques: Binding Assay, Microscopy, Immunofluorescence, Staining, Western Blot, Clone Assay, Membrane

Journal: The Journal of Biological Chemistry

Article Title: An essential signaling function of cytoplasmic NELFB is independent of RNA polymerase II pausing

doi: 10.1016/j.jbc.2023.105259

Figure Lengend Snippet: NELFB supports progrowth signaling transduction. A , Western blotting of cell lysates from MEFs depleted of NELFB by Cre-loxP. Representative results from three independent repeats. Asterisks indicate the reduced intensity of phosphorylated proteins. B – D , quantification of phosphorylation of various signaling molecules, which was normalized with total proteins from three independent experiments. Statistics was conducted using multiple t test. E , NELFB-AID and NELFC-AID MEFs were treated with PBS or 0.5 mM IAA and then harvested at different time points. Cell lysates were analyzed by Western blotting. The intensity of phosphorylation was normalized with total protein and indicated below the corresponding protein. Representative images were shown from three independent repeats. F and G , violin plot distributions of Pol II ChIP-seq reads at the TSS (−0.5 to +0.5kb) and GB (+0.5 to +2.5 kb) in NELFB-AID cells ( F ) and NELFC-AID cells ( G ) following treatment of PBS or 0.5 mM IAA for 6 h. Pol II ChIP-seq was from two independent experiments. H , NELFB-AID in MDA-MB-231 cells following IAA treatment. Cell lysates were analyzed by Western blotting. The intensity of phosphorylation was normalized with total protein Three independent clones were used for the experiment. I , NELFB-AID cell lines that stably expressed EV, WT, or MT194 were treated with IAA. Cell lysates were analyzed by Western blotting. The intensity of phosphorylation was normalized with total protein. Representative images from three independent repeats are shown. AID, auxin-induced protein degradation; ChIP-seq, chromatin immunoprecipitation sequencing; EV, empty vector; IAA, indole-3-acetic acid; MEF, mouse embryonic fibroblast; NELF, negative elongation factor.

Article Snippet: C-terminal mini-AID–tagged mouse NELFB and human NELFC were cloned into pLenti-6.3/V5-DEST-GFP (Addgene: 40125).

Techniques: Transduction, Western Blot, Phospho-proteomics, ChIP-sequencing, Clone Assay, Stable Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: The Human Integrator Complex Facilitates Transcriptional Elongation by Endonucleolytic Cleavage of Nascent Transcripts

doi: 10.1016/j.celrep.2020.107917

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal Anti-NELF-E , Santa Cruz Biotechnology , Cat# sc-377052; NA.

Techniques: Western Blot, Recombinant, Plasmid Preparation, Software

Journal: Mucosal immunology

Article Title: Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity

doi: 10.1038/s41385-021-00465-9

Figure Lengend Snippet: Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice were treated with 3 or 4% DSS in drinking water for 5 days. a–c Body weights (a), survival (b) (n = 24–28) and colon length (c) (n = 3–4) of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice after DSS treatment. d, e H&E staining (d) and histological scores (e) (n = 4) of colon sections from DSS treated mice at day 4. Scale bar, 50 μm. f, g H&E staining (f) and histological scores (g) (n = 4) of colon sections from DSS treated mice at day 8. Scale bar, 50 μm. Data are pooled from more than 3 (a, b) independent experiments and shown as mean ± SEM. Student’s t-test was performed; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Images represent at least two independent experiments (c, d, and f).

Article Snippet: RNA-mediated interference The siRNA against human NELFB and the negative control siRNA (nontargeting siRNA) were purchased from GenePharma Company.

Techniques: Staining

Journal: Mucosal immunology

Article Title: Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity

doi: 10.1038/s41385-021-00465-9

Figure Lengend Snippet: Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice were pre-treated with water or antibiotics, and continued drink with 3% DSS for 2 days. a, b TUNEL analysis (a) and quantification (b) of cell death in Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice after DSS or DSS with antibiotics treatment (n = 4–6). Scale bars, 50 μm. c, d TEM analysis (c) and quantification (d) of IEC cell death in Nelfb+/+Villin-Cre (a–d) and Nelfbfl/flVillin-Cre (e–h) mice after DSS treatment (n = 4–5). Scale bars, 5 or 10 μm. Data are pooled from at least 2 (B and D) independent experiments and shown as mean ± SEM. Student’s t-test was performed; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Images (a, c) represent at least two independent experiments.

Article Snippet: RNA-mediated interference The siRNA against human NELFB and the negative control siRNA (nontargeting siRNA) were purchased from GenePharma Company.

Techniques: TUNEL Assay

Journal: Mucosal immunology

Article Title: Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity

doi: 10.1038/s41385-021-00465-9

Figure Lengend Snippet: Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice were treated with 3% DSS in drinking water for 2 days. a, b H&E staining (a) and histological scores (b) (n = 3–5) of colon sections from DSS treated mice at day 2. Scale bars, 50 μm. c qPCR analysis of cell junction-related genes in colon tissues from DSS treated mice at day 2 (n = 5–13). d Intestinal permeability analysis of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice after DSS treatment at day 2 (n = 3). e, f FISH analysis (e) and quantification (f) of bacterial invasion in colon sections from DSS treated mice at day 2 (n = 3–5). Scale bars, 50 μm. g–i Immunofluorescent analysis (g) and quantification (h, i) of bacterial (E. coli and Clostridia). invasion in colon sections from DSS treated mice at day 2 (n = 3–6). Scale bars, 50 μm. j The experimental scheme for the procedure of antibiotics or/and DSS treatment. k, l Body weights (k) and survival (l) (n = 12–20) of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice. m, n H&E staining (m) and histological scores (m) (n = 5–6) of colon sections from antibiotics or/and DSS treated mice at day 4. Scale bars, 50 μm. All data are shown as mean ± SEM. Student’s t-test was performed; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns not significant (p > 0.05).

Article Snippet: RNA-mediated interference The siRNA against human NELFB and the negative control siRNA (nontargeting siRNA) were purchased from GenePharma Company.

Techniques: Staining, Permeability

Journal: Mucosal immunology

Article Title: Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity

doi: 10.1038/s41385-021-00465-9

Figure Lengend Snippet: a, b TEM analysis (a) and quantification (b) of IEC cell death in Nelfb+/+Villin-Cre (a, b) and Nelfbfl/flVillin-Cre (c, d) mice at stead state (n = 4–9). Scale bars, 5 or 10 μm. c, d Immunohistochemistry analysis (c) and quantification (d) of cleaved caspase 3 positive cell in colon section of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice (n = 7). Scale bar, 50 μm. e, f Immunoblotting analysis (e) and quantification (f) of cleaved caspase 1 in colon tissues of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice (n = 7–8). g, h Immunoblotting analysis (g) and quantification (h) of p-MLKL in colon tissues of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice with or without DSS treatment (n = 4–10). i, j Organoids analysis (l) and quantification (j) of TNF-α induced cell death of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice (n = 3). Scale bar, 50 μm. Data are pooled from at least two (b, d, f, h, and j) independent experiments and shown as mean ± SEM. Student’s t test was performed; *p ≤ 0.05; ns not significant (p > 0.05). Images (a, c, and i) represent at least two independent experiments.

Article Snippet: RNA-mediated interference The siRNA against human NELFB and the negative control siRNA (nontargeting siRNA) were purchased from GenePharma Company.

Techniques: Immunohistochemistry, Western Blot

Journal: Mucosal immunology

Article Title: Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity

doi: 10.1038/s41385-021-00465-9

Figure Lengend Snippet: a The volcano plot analysis of RNA-seq dataset showing the top upregulated and downregulated genes in colonic epithelia cell from Nelfbfl/flVillin-Cre mice versus Nelfb+/+Villin-Cre at stead state. b Heat map analysis of RNA-seq dataset showing the top 20 upregulated and downregulated genes in colonic epithelia cell from Nelfbfl/flVillin-Cre mice versus Nelfb+/+Villin-Cre. Red font indicates that upregulated genes are highly expressed in goblet cells and blue font indicates that downregulated genes are highly expressed in enterocytes. c qPCR analysis of the expression of representatively upregulated and downregulated genes from RNA-seq dataset in Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice (n = 5–17). d, e Immunofluorescent WGA and PAS staining (d) and quantification (e) analysis of the goblet cell in Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre (n = 6–13). Scale bars, 50 μm. Data are pooled from at least two (c, e) independent experiments and shown as mean ± SEM. Student’s t-test was performed; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns not significant (p > 0.05). Images (d) represent at least two independent experiments.

Article Snippet: RNA-mediated interference The siRNA against human NELFB and the negative control siRNA (nontargeting siRNA) were purchased from GenePharma Company.

Techniques: RNA Sequencing, Expressing, Staining

Journal: Mucosal immunology

Article Title: Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity

doi: 10.1038/s41385-021-00465-9

Figure Lengend Snippet: a Heat map of NELF-E ChIP-seq signals around TSS regions for colonic IEC-expressed genes in Nelfb+/+Villin-Cre mice at steady state. Each row indicates one gene sorted by the peak heights in Nelfb+/+Villin-Cre mice. The signals were normalized to input signals. The number of peaks in the whole genome is shown at the bottom. b Relative NELF-E ChIP-seq signals around TSS regions as (a) shown. c The genomic distribution of NELF-E ChIP-seq peaks. d GO analysis of NELF-E ChIP-seq peaks for cellular component classes and many cellular components (red font) are related to epithelia cell–cell junction. e qPCR analysis of epithelia cell junction-related genes expression in Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice (n = 6–12). Tracks of NELF-E ChIP-seq are shown for these genes. f The occupancies of NELF-E in the TSS region of cell junction-related genes were assessed by ChIP-qPCR in Nelfbfl/flVillin-Cre versus Nelfb+/+Villin-Cre mice. g, h Immunoblotting analysis (g) and quantification (h) of Cldn3 in colon tissues of Nelfb+/+Villin-Cre and Nelfbfl/flVillin-Cre mice (n = 9–12). Data are pooled from at least two (e, f, and h) independent experiments and shown as mean ± SEM. Student’s t-test was performed; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns not significant (p > 0.05). Images (g) represent at least two independent experiments.

Article Snippet: RNA-mediated interference The siRNA against human NELFB and the negative control siRNA (nontargeting siRNA) were purchased from GenePharma Company.

Techniques: ChIP-sequencing, Expressing, ChIP-qPCR, Western Blot