Fig 2: Poly-PR and TDP-43 up-regulate NEAT1 expression through distinct mechanisms.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

Fig 3: Poly-PR interacts and co-localizes with TDP-43 and nuclear TDP-43 up-regulates NEAT1 expression.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

Fig 4: TDP-43 antibody screening by immunofluorescence.HAP1 WT and TARDBP KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with a glass bottom. Cells were stained with the indicated TDP-43 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined on both channels with green and magenta dashed lines, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 12892-1-AP, MA5-32627**, A19123**, 89789**, 89718** and ab133547** which were titrated to 1/400, 1/1000, 1/900, 1/30, 1/10 and 1/700, respectively, as the signals were too weak when following the supplier’s recommendations When the concentration was not indicated by the supplier, which was the case for antibodies 80001-1-RR**, GTX630196* and ab254166**, we tested antibodies at 1/700, 1/1000 and 1/500, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: 10782-2-AP at 1/400, 12892-1-AP at 1/250, 800001-1-RR** at 1/700, 80002-1-RR** at 1/250, MAB7778* at 1/500, NBP1-92695* at 1/1000, 711051** at 1/500, MA5-27828* at 1/1000, MA5-32627** at 1/1000, A19123** at 1/900, 89789** at 1/30, 89718** at 1/10, GTX630196* at 1/1000, GTX630197* at 1/1000, ab109535** at 1/30, ab133547** at 1/700, ab190963** at 1/800, ab254166** at 1/500. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

Fig 5: TDP-43 antibody screening by Western blot.Lysates of HAP1 (WT and TARDBP KO) were prepared and 50 μg of protein were processed for Western blot with the indicated TDP-43 antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibody 80001-1-RR**, which was titrated to 1/1000 as the signal was too weak when following the supplier’s recommendations. When the concentration was not indicated by the supplier, which was the case for antibody 80002-1-RR**, we tested the antibody at 1/1000. Antibody dilution used: 10782-2-AP at 1/5000, 12892-1-AP at 1/1000, 800001-1-RR** at 1/1000, 80002-1-RR** at 1/1000, MAB7778* at 1/500, NBP1-92695* at 1/1000, 711051** at 1/1000, MA5-27828* at 1/1000, MA5-32627** at 1/1000, A19123** at 1/1000, 89789** at 1/1000, 89718** at 1/1000, GTX630196* at 1/500, GTX630197* at 1/500, ab109535** at 1/2000, ab133547** at 1/1000, ab190963** at 1/1000, ab254166** at 1/1000. Predicted band size: 45 kDa. *Monoclonal antibody, **Recombinant antibody.