Fig. 2

TDP-43 depletion but not its cytoplasmic accumulation or aggregation stimulates paraspeckle assembly in stable cell lines. a TDP-43 siRNA-mediated knockdown upregulates NEAT1_2. MCF7 cells were transfected with scrambled or TDP-43 siRNA and analysed 48 h post-transfection by qRT-PCR (n = 6). **p < 0.01 (Mann-Whitney U-test). b and c TDP-43 depletion auguments paraspeckle assembly in MCF7 cells. Quantification (b) and representative images (c) are shown. RNA-FISH with NEAT1_2 probe (c, top panels) or anti-NONO staining (c, bottom panels) were used to visualise paraspeckles; arrowheads indicate clusters of paraspeckes. The number of cells analysed is indicated in the bottom of each bar (b) (***p < 0.0001, Student’s t-test). d TDP-43 depletion enhances interaction of NEAT1_2 with core paraspeckle proteins NONO and FUS. GFP-tagged NONO or FUS was co-transfected into MCF7 cells together with scrambled siRNA or TDP-43 siRNA. NEAT1_2 and total NEAT1 were detected in GFP pull-down samples by RT-PCR. Arrowhead indicates the specific band for NEAT1_2 primer pair. e Expression of paraspeckle-regulated genes in MCF7 cells depleted of TDP-43 as measured by qRT-PCR (n = 6 or 8). *p < 0.05, ***p < 0.001 (Mann-Whitney U-test). f and g Expression of TDP-43 lacking nuclear localisation signal (TDP-43 dNLS) or TDP-43 C-terminal 25 kDa fragment (TDP-43 CT) does not affect paraspeckles or NEAT1 levels. Representative images of paraspeckles in transfected SH-SY5Y cells (f) and their quantiation (g, n = 56 and n = 41 for GFP- and TDP-43 dNLS-expressing cells respectively) are shown. Scale bars are 10 μm in all panels