Figure 6.

RAN increases TDP43 nucleus import and facilitates TDP43 binding G3BP1 mRNA.A, after treatment with actinomycin D (10 μg/ml), G3BP1 mRNA level was quantified at indicated times in control, RAN-silenced, TDP43-silenced, and both RAN and TDP43-silenced cells. The half-life of G3BP1 mRNA was analyzed by plotting degradation curves. Data are presented as the mean ± 95%CI (n = 3). B, TDP43 protein levels with knockdown of RAN were detected by Western blotting. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). C, relative enrichment of G3BP1 mRNA immunoprecipitated by anti-TDP43 or anti-IgG antibody was indicated by RT-qPCR upon knockdown of RAN. Data are presented as the mean ± SD (n = 3). D, the nucleus and cytoplasm proteins in HONE-1 and SUNE-1 cells were isolated by nuclear/cytosol fractionation assays. TDP43 distribution in the nucleus or cytoplasm was identified by Western blotting with or without RAN silencing, GAPDH as the cytoplasm marker, and Lamin B as the nucleus marker. The blots are the representation of three independent experiments. Data are presented as the mean ± SD (n = 3). E, TDP43-GFP overexpression vectors were transfected in HONE-1 and SUNE-1 cells after RAN silencing. Recognizing TDP43-GFP protein distribution (green) in HONE-1 and SUNE-1 cells after 24 h of TDP43-GFP overexpression vectors transfected, range indicator was performed to show clearer distribution of TDP43-GFP in the nucleus or cytoplasm, cell nuclei were stained with DAPI (blue). The scale bar represents 10 μm. F, the average fluorescence intensity of TDP43-GFP in the nucleus (lower) and cytoplasm (upper) of each cell was calculated. Data are presented as the mean ± SD (n = 20). ∗p < 0.05 and ∗∗p < 0.01. The significant differences were assessed using one-way ANOVA (B, D, and F), two-way ANOVA (A), and t test (C). RT-qPCR, reverse transcription quantitative real-time PCR; DAPI, 4′, 6-diamino-2-phenylindole; CI, confidence interval.