4 research outputs found
Allele-specific siM9 decreases total TDP-43 transcripts and protein levels in neuralised cells.
<p><b>A.</b> Representative Western blot image showing M337V knockdown in M337V lines. <b>B.</b> Densitometry analysis of relative TDP-43 protein normalised to GAPDH. <b>C.</b> qPCR display unchanged levels of total TDP-43 in the control lines transfected with siM9, whereas M337V lines showed a reduction. Error bars represent SEM (One way ANOVA, * P<0.05, ** P<0.01, *** P<0.001).</p
Allele-specific siRNA silences the mutant allele specifically and reduces cytoplasmic inclusions in HEK293 cells.
<p><b>A.</b> HA-TDP-43<sup>wt</sup> stably expressing HEK293 cells were co-transfected with C-terminal GFP-TDP-43<sup>wt</sup> or GFP-TDP-43<sup>M337V</sup> and siRNAs for 48 hours. Cells were fixed and stained with HA antibody (red) and DAPI (blue). GFP-TDP-43 cytosolic and nuclear inclusions of different sizes were seen. C-terminal GFP-TDP-43<sup>wt</sup> and GFP-TDP43<sup>M337V</sup> inclusions co-localized with full length HA-TDP-43<sup>wt</sup> (arrows), however some inclusions did not recruit full length HA-TDP-43<sup>wt</sup> (arrowhead). Scale bars = 20 µm. <b>B.</b> Percentage of cells with GFP aggregates. siM9 reduced the number of cells with aggregates in GFP-TDP-43<sup>M337V</sup> – expressing cells. More than 70 000 cells were counted from three independent experiments. Error bars represent SEM (Student's T-test,*** P<0.001).</p
Allele-specific siRNAs targeting TDP-43<sup>M337V</sup> mutant allele.
<p><b>A.</b> Schematic representation of TDP-43 protein containing two RNA-recognition motifs (RRM1 and RRM2), a bipartite nuclear localization signal (NLS), a nuclear export signal (NES) and a glycine-rich domain in the carboxy-terminal. The M337V mutation localization is indicated. Five allele-specific siRNAs were designed to contain mismatches at positions 9 (M9), 3 (M3), or 17 (M17); double mismatches at positions 8 and 9 (M8-9) or multiple mismatches at positions 5, 7, 10 and 16 (M5U). <b>B.</b> Representative western blot image showing the effects of allele-specific siRNA on cells transfected with GFP-TDP-43<sup>wt</sup> and GFP-TDP-43<sup>M337V</sup>. The allele-specific siM9 reduces the levels of GFP-TDP-43<sup>M337V</sup> specifically whereas GFP-TDP-43<sup>wt</sup> levels remain unchanged. FLAG-tagged protein was used as a control for transfection efficiency. <b>C.</b> Densitometry analysis of relative GFP-TDP-43 normalised to GAPDH. Mean from three independent experiments. Error bars represent standard error of the mean (SEM). (One way ANOVA, * P<0.05; *** P<0.001).</p
Allele-specific knockdown of M337V allele on neural stem cells.
<p><b>A.</b> NSCs were transfected with allele-specific siM9 and stained for TDP-43. Images were acquired using identical parameters and analysed using Metamorph software. Representative confocal immunolabeling images showing allele-specific M337V knockdown in M337V lines. The allele-specific siM9 reduces endogenous TDP-43<sup>M337V</sup> expression in all compartments (cytosolic (<b>B</b>), nuclear (<b>C</b>) and total (<b>D</b>) TDP-43) (n = 3 independent experiments. ** P<0.01 and *** P<0.001).</p