Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a Glutathione S-transferase (GST) or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were mixed with ( + ) or without (−) NSC-34 cell lysates. After the rotation at 4 °C for 5 h, the glutathione beads were washed and were subjected to 5–20% gradient gel SDS–PAGE followed by immunoblotting (IB) using indicated antibodies. In western blotting using anti-GST antibody, the large smear within the molecular weights ranging 25–46 kDa in the GF-PR lane is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to represent dimerized and/or aggregated GST-derived proteins. b NSC-34 cells overexpressing EGFP-FLAG-PR100 (EGFP-F-PR100) (green) together with HA-tagged hnRNPF, hnRNPH1, or hnRNPM were fixed and were immunostained with HA (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 10 μm. The rightmost panel shows the profile image of fluorescence intensities on the lines of EGFP-FLAG-PR100 and hnRNPs-merged image. Arrows and arrowheads indicate the localization of nucleoplasm-localizing EGFP-FLAG-PR100 and the border of nucleolus-localizing EGFP-FLAG-PR100, respectively. c Lysates of NSC-34 cells overexpressing HA-tagged hnRNPF, hnRNPH1, or hnRNPM and GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with ( + ) or without (−) 20 μg/mL RNase A. After the incubation, cell lysates were mixed with GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and were subjected to immunoblotting (IB) using indicated antibodies. d , e NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG1 (Cont.) or the FLAG antibody. Precipitates were then used for RNA immunoprecipitation (RIP) assay ( d ) and dot blotting analysis with the FLAG antibody ( e ). Reverse transcription (RT) (−) was used as negative control to monitor the PCR amplification from genomic DNA. f NSC-34 cells overexpressing EGFP or EGFP-FLAG-PR100 (green) were fixed and stained with the 500 nM Quasar 570-labeled NEAT1 Stellaris probe (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. Arrowheads and arrow correspond to paraspeckles. The rightmost panel shows the profile image of fluorescence intensities on the lines of EGFP-FLAG-PR100 and NEAT1-merged image in magnified image. Arrowhead and arrow indicate the co-localization of NEAT1 with nucleoplasm-localizing EGFP-FLAG-PR100 and the nucleolus-localizing EGFP-FLAG-PR100, respectively

Article Snippet: Hs03453534_s1 detects both NEAT1_1 and NEAT1_2.

Techniques: SDS Page, Western Blot, Derivative Assay, Staining, Fluorescence, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation, RNA Immunoprecipitation, Reverse Transcription, Negative Control, Amplification, Labeling

Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a The number of EGFP- or EGFP-FLAG-PR100 (EGFP-F-PR100)-expressing paraspeckle-positive NSC34 cells in RNA FISH assay was counted (100 cells/count). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by the unpaired t -test. b, c NSC-34 cells were infected with indicated adenovirus vectors at a multiplicity of infection (MOI) of 800. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( b ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( c ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Dunnett’s multi comparisons test. d , e Primary cultured cerebral cortical neurons (PCNs) were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–200. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 120 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( d ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( e ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. f NSC-34 cells were transfected with the NEAT1 -promoter (+) or NEAT1 -promoterless (−) luciferase vector together with the pEF1-Myc/His-vec (−) or the pEF1-FLAG-PR100 (+). At 48 h after the transfection, the luciferase activity was measured. The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. g , h NSC-34 cells were infected with adenovirus encoding LacZ or mouse NEAT1_1 at an MOI of 1. Cells were also co-infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 200 together with adenovirus encoding LacZ (−) or Cre-recombinase (+) at an MOI of 40. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( g ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( h ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. i , j NSC-34 cells were transfected with 0.2 μg/well of the pCMV-GFP or -mouse NEAT1_2 on 6-well plate. After the transfection, NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at an MOI of 200. Total adenoviruses infected were adjusted at an MOI of 400 with adenovirus encoding LacZ. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( i ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( j ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. k , l NSC-34 cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1, Nfkb1, and Nr4a1 was performed ( k ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( l ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by the unpaired t -test. m–o HeLa cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800. At 48 h after the infection, the cell viability was detected by WST-8 assay ( m ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( n ). The quantitative real-time PCR analysis of NEAT1, CCL5, CXCL8, SH3PXD2A, and TSHZ2 was performed ( o ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by the unpaired t -test

Article Snippet: Hs03453534_s1 detects both NEAT1_1 and NEAT1_2.

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Cell Culture, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a , b NSC-34 cells were transfected with 0.25 μg/well of the U6-sgRNA(MS2)_EF1a-MS2-P65-HSF1 vector (Vec) or the guide RNA sequence against NEAT1 promoter-containing U6-sgRNA(MS2)_EF1a-MS2-P65-HSF1 vector (NEAT1) in association with 0.75 μg/well of the pcDNA3 vector (Vec) or the pAC152-dual-dCas9VP64-sgExpression vector (dCas9-VP64) on 6-well plate. At 72 h after the transfection, the quantitative real-time PCR analysis of NEAT1 was performed ( a ). The cell viability was detected by WST-8 assay ( b ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. c – e NSC-34 cells were infected with adenovirus encoding mouse NEAT1_1 or FLAG-PR100 at an MOI of 400 together with adenovirus encoding LacZ or Cre-recombinase at an MOI of 40. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( c ). The cell viability was detected by WST-8 assay ( d ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( e) . The data are presented as means ± SD ( N = 3). Statistical analysis for data obtained from ( c ) NEAT1_2 and ( d ) was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. Statistical analysis for data obtained from ( c ) NEAT1_1 & NEAT1_2 was conducted by the unpaired t -test

Article Snippet: Hs03453534_s1 detects both NEAT1_1 and NEAT1_2.

Techniques: Transfection, Plasmid Preparation, Sequencing, Real-time Polymerase Chain Reaction, Infection

Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the cell lysates were subjected to immunoblotting (IB) and dot blotting analysis using indicated antibodies. Intensities of immunodetected signals of endogenous hnRNPM were densitometrically examined with an ImageJ software. b , c NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the quantitative real-time PCR analysis of hnRNPM was performed ( b ). The cell lysates were subjected to dot blotting analysis using indicated antibodies ( c ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. d , e NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( d ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( e ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. f , g NSC-34 cells were transfected with 0.2 μg/well of the pCMV-GFP or -mouse NEAT1_2 on 6-well plate. After the transfection, NSC-34 cells were infected with adenovirus encoding LacZ or hnRNPM at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( f ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( g ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. h , i NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the cell viability was detected by WST-8 assay ( h ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( i ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test

Article Snippet: Hs03453534_s1 detects both NEAT1_1 and NEAT1_2.

Techniques: Infection, Western Blot, Software, Real-time Polymerase Chain Reaction, Transfection

Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After rotation at 4 °C overnight, the glutathione beads were washed and were subjected to immunoblotting (IB) using indicated antibodies. In western blotting using anti-GST antibody, the large smear within the molecular weights ranging 25–46 kDa in the GF-PR lane is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to represent dimerized and/or aggregated GST-derived proteins. b Lysates of NSC-34 cells overexpressing HA-tagged TDP-43 and GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with (+) or without (−) 20 μg/mL RNase A. After the incubation, the cell lysates were mixed with recombinant GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and were subjected to immunoblotting (IB) using indicated antibodies. c NSC-34 cells overexpressing EGFP-FLAG-PR100 (EGFP-F-PR100) (green) were fixed and were immunostained with TDP-43 antibody (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. The rightmost panel shows the profile image of fluorescence intensities on the line of EGFP-FLAG-PR100 and TDP-43-merged image. Arrows and arrowheads indicate the localization of nucleoplasm-localizing EGFP-FLAG-PR100 and the border of nucleolus-localizing EGFP-FLAG-PR100, respectively. d , e NSC-34 cells were infected with adenovirus encoding LacZ or TDP-43 at an MOI of 800. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( d ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( e ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by the unpaired t-test. f NSC-34 cells were transfected with the NEAT1 -promoter (+) or NEAT1 -promoterless (−) luciferase vector together with the pEF1-Myc/His-vec (−) or the pEF1-TDP-43 (+). At 48 h after the transfection, the luciferase activity was measured. The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. g – i NSC-34 cells were infected with adenovirus encoding LacZ or TDP-43 derivatives at an MOI of 800. At 48 h after the infection, the cell viability was detected by WST-8 assay ( g ). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies ( h ). CTF35, C-terminal fragment 35 kDa. The quantitative real-time PCR analysis of NEAT1 was performed ( i ). The data are presented as means ± SD ( N = 3). Statistical analysis for ( g ) was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. Statistical analysis for ( i ) was conducted by one-way ANOVA followed by the Dunnett’s multi comparisons test

Article Snippet: Hs03453534_s1 detects both NEAT1_1 and NEAT1_2.

Techniques: Western Blot, Derivative Assay, Incubation, Recombinant, Staining, Fluorescence, Infection, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Cell Death & Disease

Article Title: C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

doi: 10.1038/s41419-019-1983-5

Figure Lengend Snippet: a, b NSC-34 cells were transfected with the 5 nM control siRNA (−) or the TDP-43 siRNA (+). At 18 h after the transfection, cells were infected with adenovirus encoding LacZ (−) or FLAG-PR100 (+) at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( a ). The cell lysates were subjected to immunoblotting (IB) and dot blotting analysis using indicated antibodies ( b ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. c , d NSC-34 cells were infected with adenovirus encoding LacZ (−) or TDP-43 (+) at an MOI of 400. Cells were also co-infected with adenovirus encoding LacZ (−) or FLAG-PR100 (+) at an MOI of 200. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed ( c ). The cell lysates were subjected to immunoblotting (IB) and dot blotting analysis using indicated antibodies ( d ). The data are presented as means ± SD ( N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test

Article Snippet: Hs03453534_s1 detects both NEAT1_1 and NEAT1_2.

Techniques: Transfection, Control, Infection, Real-time Polymerase Chain Reaction, Western Blot