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: siRNAs against mouse hnRNPF, hnRNPH1, and TDP-43 and non-targeting control siRNA were purchased from RNAi Co., Ltd. (Tokyo, Japan).

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 , b NSC-34 cells were infected with adenovirus encoding LacZ or hnRNPF at an MOI of 200. Cells were also co-infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 400. At 48 h after the infection, the cell lysates were subjected to immunoblotting (IB) and dot blotting analysis using indicated antibodies ( a ). Intensities of immunodetected signals of endogenous hnRNPH1 were densitometrically examined with an ImageJ software ( b ). 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. c , d NSC-34 cells were co-transfected with the 5 nM control, hnRNPF, and hnRNPH1 siRNAs. To keep the total concentration of siRNA at 10 nM, an appropriate amount of control siRNA was added for each transfection. At 60 h after the transfection, the cell viability was detected by WST-8 assay ( c ). The cell lysates were subjected to immunoblotting (IB) 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: siRNAs against mouse hnRNPF, hnRNPH1, and TDP-43 and non-targeting control siRNA were purchased from RNAi Co., Ltd. (Tokyo, Japan).

Techniques: Infection, Western Blot, Software, Transfection, Control, Concentration Assay