guide rna plasmids human nsmf  (Addgene inc)

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