Cross-regulation between TDP-43 and paraspeckles promotes pluripotency-differentiation transition
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additional supplementary data: - Sanger sequencing and confirmatory gel electrophoresis confirming the CRISPR-Cas9 edited human and mouse embryonic stem cells presented in the manuscript under review. - additional supplementary data: raw unprocessed confocal images presented in Fig2 of the manuscript under review:
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RNA-FISH - immunofluorescence was performed as previously described (Kawaguchi et al., 2015; West et al., 2016). The RNA probes were synthesized using T7 or SP6 RNA polymerase and a digoxigenin (DIG) RNA labeling kit (11175025910, Roche). Linearized plasmids (1 g) containing a NEAT1 fragment (NEAT1 1-1000 nt) were used as templates. Cells were grown on coverslips (Matsunami; micro cover glass; 18 mm round; thickness, 0.16–0.19 mm) and treated with 5 uM proteasome inhibitor (MG132, Sigma-Aldrich) for 6 h to enhance NEAT1 expression (Hirose et al., 2014). Then cells were fixed with 4% paraformaldehyde/PBS at room temperature for 10 min, washed using 1× PBS, permeabilized with 0.5% Triton X-100 (9002-93-1, Sigma-Aldrich)/PBS for 5 min, and washed three times with 1× PBS. Subsequently, the cells were dipped in 100% ethanol for 5 min and then dried. Dehydrated coverslips were incubated with pre-hybridization solution (50% formamide, 1× Denhardt’s salt, 2× SSC, 100 mM EDTA, 100 ug/ml yeast tRNA, and 0.01% Tween 20) at 55°C for 1 h and then incubated with hybridization solution (as above with 5% Dextran sulfate) containing the DIG-labeled RNA probes (final concentration: 100 ng/coverslip) at 55°C o/n. After hybridization, the coverslips were washed twice with pre-warmed wash buffer (50% formamide, 2× SSC, and 0.1% Tween 20) at 55°C for 15 min. Then, excess RNA probes were digested with 10 ug/ml RNase A (9001-99-4, Nacalai Tesque) in NTET buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 500 mM NaCl, and 0.1% Tween 20) at 37°C for 30 min. The coverslips were washed with buffer (2× SSC and 0.01% Tween 20) at 55°C for 15 min and washed twice with a second buffer (0.1× SSC and 0.01% Tween 20) at 55°C for 15 min. The coverslips were subsequently washed with TBST (1× TBS containing 0.1% Tween 20) and incubated with 1× blocking solution (Blocking reagent [Roche] diluted with TBST) for blocking at RT for 1 h. Then, the coverslips were incubated with primary antibodies, Anti-TDP-43 rabbit polyclonal antibody (Proteintech, 1:100), anti-PSPC1 rabbit polyclonal antibody (1:1000; Naganuma et al., 2012) in 1× blocking solution at room temperature for 1 h, washed three times with TBST for 5 min, and incubated with secondary antibodies, anti-mouse IgG Alexa-488, and anti-rabbit IgG, Alexa-568 (both Thermo Fisher Scientific]) in 1× blocking solution at RT for 30 min, and washed three times with TBST for 5 min. The coverslips were mounted with VECTASHIELD Hard Set Mounting Medium with DAPI (Vector). Confocal images were acquired using a confocal laser scanning microscope FV1000D (Olympus). Cells were automatically counted using Fiji software. First, nuclei or paraspeckles were segmented using either DAPI or FISH staining to identify areas of interest. Then gausian blur (1.2) was applied and threshold for each object was calculated using Otsu’s method. Mean intensity values or correlation coefficients were measured for the respective areas.