Additional file 3: of Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

Figure S3. Accumulation of dsRNA (a) and increased levels of p-eIF2α (b) in MCF7 cells depleted of TDP-43. Cells were analysed 48 h post-transfection. Scale bars, 100 μm and 10 μm for general plane and close-up panels respectively. (DOCX 663 kb

Additional file 5: of Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

Figure S5. Differentiation of human neuroblastoma cells into neuron-like cells does not lead to the loss of paraspeckles. Differentiated SH-SY5Y cells develop extensive neurite network and are uniformly positive for a neuronal marker Tuj 1 (left panel) but preserve their ability to form paraspeckles (right panel). SH-SY5Y cells were induced to differentiate into neuron-like cells using retinoic acid/BDNF and analysed 6 days into differentiation by immunocytochemitry and NEAT1_2 RNA-FISH. Representative images are shown. Scale bars, 100 μm (left panel) and 10 μm (right panel). (DOCX 175 kb

Additional file 1: of Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

Figure S1. The effect of TDP-43 dysfunction on paraspeckles, speckles and paraspeckle proteins in MCF7 and SH-SY5Y cells. a and b TDP-43 siRNA-mediated knockdown upregulates NEAT1_2 (a) and enhances paraspeckle assembly (b) in SH-SY5Y cells. Cells were transfected with scrambled siRNA or Silencer® TDP-43 siRNA and analysed by qRT-PCR (n = 6). Mean number of paraspeckles per cell was also quantified using NEAT1_2 RNA FISH. **p < 0.01, ***p < 0.001 (Mann-Whitney U-test in a and Student’s t-test in b). c and d Downregulation of TDP-43 using an esiRNA (endoribonuclease-prepared MISSION® esiRNA, c) or shRNA (d) stimulates paraspeckle assembly. The efficiency of knockdown was analysed by TDP-43 immunocytochemistry, and paraspeckle assembly – by NEAT1_2 RNA-FISH. Representative images are shown. Scale bars, 100 μm for left panels and 10 μm for right panels. e Speckles visualised by MALAT1 RNA-FISH are not affected by TDP-43 knockdown. Representative images are shown. Scale bar, 10 μm. f Levels of core paraspeckle proteins NONO, SFPQ and FUS are not affected by TDP-43 knockdown in SH-SY5Y cells. Representative Western blots are shown. g Sequences and positions of gRNAs used for disrupting the NLS of the endogenous TDP-43 protein by CRISPR/Cas9-mediated editing. Two combinations of upstream and downstream gRNA sequences within TARDBP gene selected to disrupt the NLS are shown. The sequence encoding for the NLS is given in blue and PAM sites are boxed. h Transient transfection of two combinations of plasmids encoding upstream and downstream gRNAs for targeting the NLS of TDP-43 results in partial redistribution of endogenous TDP-43 but does not lead to enhanced paraspeckle formation. Cells were analysed 72 h post-transfection. Representative images are shown, asterisks indicate cells with cytoplasmic TDP-43 redistribution. Scale bar, 10 μm. In a-f, cells were analysed 48 h post-transfection. (DOCX 517 kb

Additional file 6: Figure S6. of Insulin and IGF1 signalling pathways in human astrocytes in vitro and in vivo; characterisation, subcellular localisation and modulation of the receptors

Reduced IGF1R but not downstream signalling in human astrocytes derived from temporal lobe resections using MAB391. Human astrocytes treated with MAB391 for 24 h. A) Immunoblots for IGF1R, pAkt, total Akt, pP44/42 MAPK and total p44/42 MAPK. α-tubulin was used as a loading control for blots. Molecular weight markers are indicated (kDa). (B) Bar charts showing quantification of blots, pAkt was normalised to Akt/tubulin. students unpaired t-test, **** p < 0.0001. (TIFF 1949 kb

Additional file 1: Figure S1. of Immune response in peripheral axons delays disease progression in SOD1G93A mice

Normalized frequency histograms of the α values computed from all the acquired RP-CARS z-stacks in the four conditions: Ntg vs. SOD1G93A and C57 vs. 129Sv. Figure S2. GFAP and P-ERK expression is reduced in the sciatic nerve of 129Sv_Ntg. Figure S3. H2-K_D (MhcI; red), and β2m (blue) mRNA levels from laser captured MNs of C57Ntg versus 129SvNtg mice. H2-K_D (red), Lmp7 (green) and β2m (blue) and Tap1 (black) longitudinal mRNA levels from laser captured MNs of C57SOD1G93A versus Ntg littermates during the progression of the disease. MHCI immunohistochemistry shows an increased and widely diffuse expression in the ventral grey and white matter of the lumbar spinal cord of C57SOD1G93A mice at disease onset compared to Ntg littermates β2M (green) and LMP7 (red) colocalization in a subset of lumbar spinal MNs (blue, NT) of C57SOD1G93A mice (inset) at the onset compared to Ntg littermates (scale bar: 50 μm; inset scale bar: 20 μm). Representative western blot of LMP7 and β2M expression in protein extracts from longitudinally dissected lumbar ventral spinal cord of C57SOD1G93A mice at the disease onset versus Ntg littermates. Figure S4. β2M is activated in the peripheral nerves of SOD1G93A mice. Figure S5. Real-time PCR for CD8+ co-receptor transcript in the spinal cord of 129SvSOD1G93A mice and Ntg littermates at disease onset. Figure S6. Confocal Z-stack orthogonal projection of a longitudinal section of sciatic nerve of C57SOD1G93A mice showing the co-localization of signals between S100beta (red) and Iba1(green), dapi (blue). RT-qPCR for Ym1, Tnfα, Il6 and Ly6c co-receptor transcript in the sciatic nerve of C57SOD1G93A mice compared to Ntg littermates at disease onset. (PDF 1222 kb

Additional file 1: Figures S1–S4. of A data-driven approach links microglia to pathology and prognosis in amyotrophic lateral sclerosis

Contain plots of pathology counts in ALS-motor neurons and details of the WGCNA analysis used to derive network modules. (PDF 723 kb

Distribution of issues addressed and an example region.

<p>(Top Panel) Issues for GRCh37, GRCh37.p1, and GRCh37.p2, broken down by type. Issue types are: Clone Problem: The issue is contained within a single clone. This may be a single nucleotide difference or a clone mis-assembly. Path Problem: There is evidence that the tiling path within a given region is incorrect and we will need to update the path. GRC Housekeeping: Changes use to help regularize the tiling path. Missing Sequence: Sequence that we can’t yet place on the assembly. Mapping studies are ongoing to help place these sequences. Variation: There is evidence to suggest that complex variation is complicating a region and an alternate allele may need to be produced. Gap: The issue concerns filling a gap. Unknown: Issue is still under investigation for classification. (Bottom Panel) Details for issue HG-2, a Path Problem. The representation in NCBI36 was a mixed haplotype. The tiling paths for NCBI36 and GRCh37 are shown. Blue clones are anchor clones that are in NCBI36, the GRCh37 chr4 path, and the GRCh37 alternate locus path. Red clones represent the UGT2B17 insertion path and dark gray clones represent the UGT2B17 deletion path. The light gray clone was not used in NCBI36, but was used in GRCh37 to complete the alternate locus.</p

Assembly representation for GRCh37.p3.

<p>The top panel shows an ideogram representation of the human genome. The primary assembly unit contains sequences for the non-redundant haploid assembly; this includes the scaffolds that make up the chromosome sequence as well as unplaced and unlocalized scaffolds that are thought to represent novel sequence (not shown in this picture). Alternate loci and patches are placed in separate assembly units to facilitate annotation. Note the seven alternate scaffolds in the MHC region are all placed in different assembly units, as they all represent different representations of the same sequences. Other alternate loci can be added to these assembly units at the next major release if they don’t overlap the existing alternates. All patches are placed in the PATCHES assembly unit and minor releases are cumulative such that the latest minor release will contain all patches. The red triangle, yellow circles, and blue circles represent regions that contain additional sequences that are not given actual chromosome coordinates, but rather are given a chromosome context via alignment to the primary assembly. The red triangles represent regions’ alternate loci; these are sequences that provide an additional tiling path to the one given in the chromosome representation and are essential for representing structurally complex loci. The circles represent patch sequences; these are minor updates made to the assembly outside of the major build cycle. Yellow circles represent “fix” patches: regions of the chromosome assembly that will change with the next major assembly update. Blue circles represent “novel” patches: these are sequences that represent new alternate loci in the next major assembly update. Unlocalized and unplaced sequences are not represented in this figure. Sequences within the assembly are placed within containers known as assembly units. Note: a region can point to more than one type of extra chromosomal sequence; for example, a region could point to an alternate locus and to a fix or novel patch.</p