Limiting factors in stochastic IFNβ gene expression.

<p>(A) Different L929 stable transfectants were induced by tetracycline (Tet) for 24 h, followed by SeV infection for 9 h. RNA ISH experiments were carried out to detect the IFNβ mRNA. (B and D) Histograms showing the percentage (mean ± standard deviation) of cells expressing IFNβ from three independent ISH experiments. At least 400 cells were blindly counted and scored for each category. (C) L929 stable transfectant was transiently transfected with expression vectors encoding either <i>GFP</i> (control), <i>RIG-I</i>, or <i>Trim25</i>, then stimulated with tetracycline for 24 h. Cells were then infected with SeV for 6 h, followed by RNA ISH to detect IFNβ mRNA. pt, pt-REX-DEST30.</p

Stochastic IFN and virus-inducible gene expression.

<p>(A) Stochastic IFNβ gene expression detected by ISH using a digoxygenin-labeled IFNβ RNA probe. (B) Percentage of IFNβ-producing cells at different times after SeV infection. (C) Mouse IFNα gene expression in primary MEFs detected by ISH using a digoxygenin-labeled IFNα4 probe. (D) qPCR analysis illustrating the expression levels of different virus-inducible genes in sorted IFNβ/YFP MEFs.</p

Poly I∶C–induced stochastic IFNβ expression depends on the amounts of poly I∶C and MDA5.

<p>(A) IFNβ/YFP homozygous MEF cells were electroporated with Cy5-labeled poly I∶C, and FACS analysis was carried out 8 h after the electroporation to assay the strength of Cy5 and YFP. The top left panel shows untransfected MEF cells, and the bottom left panel shows the electroporated MEF cells. As indicated by arrows, the two panels to the right represent the “poly I∶C high” and “poly I∶C low” populations, respectively. Data shown are representative of at least three independent experiments. Numbers represent relative percentages. (B) L929-MDA5 or L929-RIG-I stable transfectants were stimulated with tetracycline (Tet) for 24 h followed by transient transfection with poly I∶C. 6 h after transfection, cells were fixed, followed by RNA ISH to detect IFNβ mRNA. (C) Bar plots representing the percentage (mean ± standard deviation) of cells expressing IFNβ from three independent ISH experiments performed as in (B). At least 400 cells were blindly counted and scored for each category. (D) IFNβ/YFP primary MEFs were fixed 8 h after poly I∶C stimulation. Intracellular staining using MDA5 antibody and FACS analysis were carried out to assay the correlation between the expression levels of IFNβ and MDA5. Data shown are representative of at least three independent experiments. Numbers represent relative percentages. Iso-Ctrl, isotype control.</p

Viral transcription and/or replication are more efficient in IFNβ-producing cells.

<p>(A) qPCR analysis illustrating the relative abundance of viral NP, matrix (M), and L polymerase protein (L) mRNA in sorted IFNβ/YFP MEFs. (B) Western blots showing cytoplasmic distribution of SeV NP protein present in IFNβ-producing and nonproducing cells. (C) qPCR analysis illustrating the relative abundance of SeV DI genome (upper panel), and semi-qRT-PCR analysis illustrating the relative abundance of SeV genomic RNA (lower panel) in sorted IFNβ/YFP MEFs. Reverse transcriptase PCR was carried out to detect viral genomic RNA and host cell β-actin mRNA (control) using gene-specific primers. After 35 cycles (SeV genomic RNA) or 26 cycles (β-actin) of amplification, PCR products were run on a 2% agarose gel. (D) Intracellular staining using SeV antibody and FACS analysis were carried out to determine the correlation between SeV infection and IFNβ expression in IFNβ/YFP homozygous MEFs. IB, immunoblot.</p

IRF7 is the significant limiting factor in stochastic type I IFN gene expression.

<p>(A) Different L929 stable transfectants were induced by tetracycline (Tet) for 24 h, followed by SeV infection for 9 h. RNA ISH experiments were carried out to detect IFNβ mRNA. (B and D) Histograms showing the percentage (mean ± standard deviation) of cells expressing IFNβ from three independent ISH experiments. At least 400 cells were blindly counted and scored for each category. (C) RNA ISH experiments were carried out to detect IFNβ mRNA in wild-type (W.T.) or 4E-BP double-knockout (DKO) MEFs infected by SeV for 9 h.</p

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^wt stably expressing HEK293 cells were co-transfected with C-terminal GFP-TDP-43^wt or GFP-TDP-43^M337V 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^wt and GFP-TDP43^M337V inclusions co-localized with full length HA-TDP-43^wt (arrows), however some inclusions did not recruit full length HA-TDP-43^wt (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^M337V – 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^M337V 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^wt and GFP-TDP-43^M337V. The allele-specific siM9 reduces the levels of GFP-TDP-43^M337V specifically whereas GFP-TDP-43^wt 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^M337V 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