Journal: Nucleic Acids Research

Article Title: NSMF promotes the replication stress-induced DNA damage response for genome maintenance

doi: 10.1093/nar/gkab311

Figure Lengend Snippet: NSMF localizes to DNA lesions via the central residues of NSMF. ( A ) The kinetics of GFP-NSMF and mCherry-CDC5L recruitment to DNA lesions were examined by live cell imaging. The relative signal intensities are quantified and summarized in the bottom panel. The highest intensity on any lesion was set at 100% for each cell, and the recruitment kinetics were plotted. The average intensity for ≥10 cells for each condition is presented graphically. Data represent the mean ± SD. ( B ) HeLa cells were treated with 1 μM CPT for 2 h or with 2 mM HU for 16 h and then harvested as whole-cell extracts (WCEs) or fractionated into soluble or chromatin-bound protein fractions. Each fraction was analyzed by western blot with the indicated antibodies. ( C ) HeLa cells transfected with GFP-NSMF expression plasmids were treated with 2 mM HU for 16 h and then incubated in fresh media for 3 h. Colocalization of GFP-NSMF and RPA2 was determined by immunofluorescence with an RPA2 antibody. Magnified insets show colocalization of NSMF and RPA2 foci. ( D ) HeLa cells were transfected with expression plasmids for GFP-NSMF WT or GFP-NMSF deletion mutants. After 24 h, the cells were laser microirradiated, and recruitment of GFP-NSMF proteins to DNA lesions was examined by live cell imaging. Relative intensities of the lesions were quantified, and the recruitment kinetics are plotted in the right panel. The average intensity for ≥10 cells for each condition is presented graphically. Data represent the mean ±SEM of two independent experiments. ( E ) A schematic diagram of NSMF WT and the NSMF A3A4 mutant. Black boxes indicate the NLS. ( F ) HeLa cells were transfected with GFP-NSMF WT or the GFP-NSMF A3A4 mutant. The percentages of GFP-NSMF cells recruited to DNA lesions are presented graphically. More than 10 cells were analyzed for each condition. Data represent the mean ± SEM of two independent experiments. ( G ) Hela cells were transfected with control or CDC5L siRNA. After 24 h, the cells were transfected with GFP-NSMF and incubated for another 24 h. The cells were then laser microirradiated, and recruitment of the GFP-NSMF proteins to DNA lesions was examined by live cell imaging. The relative intensities of the lesions were quantified. The recruitment kinetics are plotted on the bottom panel. The average intensity of ≥ 20 cells for each condition is presented graphically. Data represent the mean ± SEM of two independent experiments.

Article Snippet: Guide RNA plasmids for human NSMF were generated by cloning guide sequences into pX330 (plasmid number 42230; Addgene).

Techniques: Live Cell Imaging, Western Blot, Transfection, Expressing, Incubation, Immunofluorescence, Mutagenesis

Journal: Nucleic Acids Research

Article Title: NSMF promotes the replication stress-induced DNA damage response for genome maintenance

doi: 10.1093/nar/gkab311

Figure Lengend Snippet: NSMF directs recruitment of ATRIP-ATR to DNA lesions by mediating the interaction between RPA2 and ATRIP-ATR. ( A ) NSMF WT and KO HeLa cells were transfected with GFP-ATRIP expression plasmids. After 24 h, the cells were microirradiated, and recruitment of GFP-ATRIP to DNA lesions was examined by live cell imaging. ( B ) NSMF WT and KO HeLa cells were treated with or without 2 mM HU for 16 h. The cells were fractionated, and the chromatin-bound protein fraction was subjected to western blot with the indicated antibodies. ( C ) Cotransfection of GFP-ATRIP with the mCherry-vec, mCherry-NSMF-WT or mCherry-NSMF-D4 mutant plasmid in NSMF KO#1 and KO#2 HeLa cells. After 24 h, the transfected cells were microirradiated, and recruitment of ATRIP and NSMF to DNA lesions was examined by live cell imaging. ( D and E ) HeLa-cell lysates were immunoprecipitated with anti-IgG, anti-ATRIP or anti-RPA2 antibodies. Endogenous interactions between NSMF and ATRIP or RPA2 were determined by western blot with the indicated antibodies. ( F and G ) HEK293T cells were cotransfected with a Flag-NSMF–deletion mutant and either GFP-ATRIP or Myc-RPA2. Immunoprecipitation using an anti-FLAG antibody and western blot with the indicated antibodies were performed to identify the NSMF regions that interact with ATRIP (F) or RPA2 (G). ( H ) HEK293T cells were transfected with GFP-ATRIP or Myc-RPA2 and either FLAG-NSMF WT or FLAG-NSMF D2. Immunoprecipitation with an anti-FLAG antibody and western blot with the indicated antibodies were performed. ( I ) Endogenous RPA2 in NSMF WT and KO HeLa-cell lysates was immunoprecipitated with anti-RPA2 antibody, and western blot with the indicated antibodies was performed.

Article Snippet: Guide RNA plasmids for human NSMF were generated by cloning guide sequences into pX330 (plasmid number 42230; Addgene).

Techniques: Transfection, Expressing, Live Cell Imaging, Western Blot, Cotransfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: RNA polymerase II pausing factor NELF in CD8 + T cells promotes antitumor immunity

doi: 10.1038/s41467-022-29869-2

Figure Lengend Snippet: a , b Percentages of CD62L + CD45RA + cells within CD4 + ( a ) and CD8 + ( b ) populations of 28z or 28z-hNELFB CAR-T cells after 10 days of in vitro expansion, n = 4/group. c Survival curves of Raji tumor-bearing mice receiving PBS or mock T cells, CD19 CAR-28z T cells, or CD19 CAR-28z-hNELFB T cells. d Model of enhancer–promoter looping mediated by NELFB, Pol II, and TCF1. Mean differences were compared using Student’s t -test. Log-rank (Mantel–Cox) tests were used for survival analyses. Source data are provided as a Source Data file.

Article Snippet: The human NELFB gene coding sequence was inserted downstream of the CAR19-28z-encoding sequence, with the P2A cleavage sequence in between (Gene Universal, Inc.).

Techniques: In Vitro

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: Molecular cell

Article Title: NELF regulates a promoter-proximal step distinct from RNA Pol II pause-release

doi: 10.1016/j.molcel.2020.02.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies TH1L (D5G6W) Rabbit mAb (NELFC/D) Cell Signaling Cell Signaling Technology Cat# 12265, RRID:AB_2797862 Recombinant Anti-NELFe antibody [ {"type":"entrez-protein","attrs":{"text":"EPR11600","term_id":"523377171","term_text":"EPR11600"}} EPR11600 ] Abcam Abcam Cat# ab170104, RRID:AB_2827280 Purified Mouse Anti-DSIF Clone 17/DSIF (SPT5) BD Biosciences BD Biosciences Cat# 611107, RRID:AB_398420 Anti-RNA polymerase II subunit B1 (phospho CTD Ser-2), clone 3E10 Millipore Millipore Cat# 04-1571, RRID:AB_11212363 Anti-RNA polymerase II subunit B1 (phospho-CTD Ser-5), clone 3E8 Millipore Millipore Cat# 04-1572, RRID:AB_10615822 NCBP1/CBP80 Antibody Bethyl Bethyl Cat# A301-793A, RRID:AB_1211224 XRN2 antibody Bethyl Bethyl Cat# A301-103A, RRID:AB_2218876 DCP2 antibody Bethyl Bethyl Cat# A302-597A, RRID:AB_10555903 HSP 90alpha/beta (F-8) antibody Santa Cruz Biotechnology Santa Cruz Biotechnology Cat# sc-13119, RRID:AB_675659 Anti-Tubulin, beta E7 DSHB DSHB Cat# E7, RRID:AB_528499 Rabbit anti-H3K4me3 serum Shilatifard Laboratory Chemicals, Peptides, and Recombinant Proteins Flavopiridol Cayman Cat# 10009197 3-indole-acetic acid sodium salt Abcam Cat# ab146403 NVP-2 MedChemExpress Cat# HY-12214A Paraformaldehyde (Electron Microscopy Sciences) Fisher Scientific Cat# 50-980-487 Biotin-11-ATP PerkinElmer Cat# NEL544001EA Biotin-11-CTP PerkinElmer Cat# NEL542001EA Biotin-11-GTP PerkinElmer Cat# NEL545001EA Biotin-11-UTP PerkinElmer Cat# NEL543001EA RNA 5’ Pyrophosphohydrolase (RppH) NEB Cat# M0356S PNK NEB Cat# M0201L T4 RNA ligase I NEB Cat# M0204L Phusion Hot Start II DNA polymerase ThermoFisher Cat# F549S Terminator 5’-triphosphate dependent exonuclease Lucigen Cat# {"type":"entrez-protein","attrs":{"text":"TER51020","term_id":"1599868412","term_text":"TER51020"}} TER51020 Quick CIP NEB Cat# M0525S Proteinase K Roche Cat# 3115828001 Dynabeads Protein G ThermoFisher Cat# 10003D Protein A/G PLUS-Agarose Santa Cruz Biotechnology Cat# sc-2003 Dynabeads Streptavidin M-280 ThermoFisher Cat# 11205D 2% Agarose, PippinHT, 100-600 bp.10/pkg.

Techniques: Recombinant, Purification, Electron Microscopy, Plasmid Preparation, Software