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Results: 11

1.
Figure 1

Figure 1. TDP-43 constructs: Diagram showing the TDP-43 constructs used in this report.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

EGFP, Enhanced Green Fluorescent Protein; RRM, RNA recognition motif; GRR, Glycine rich domain; NLS, nuclear localization signal; NES, nuclear export signal.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
2.
Figure 5

Figure 5. Endogenous TDP-43 forms inclusions after treatment with arsenite (0.5 mM, 1 hr, arrows, BE-M17 cells), and the inclusions are positive for TIA-1 (red).. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

TDP-43 inclusions are suppressed by co-treatment with cycloheximide (50 µg/ml, 1 hr). Inclusions positive for TDP-43 or TIA-1 are not evident under basal (Control) conditions. Scale bar: 10 µm.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
3.
Figure 8

Figure 8. ALS-linked TDP-43 mutations increase aggregation in response to arsenite-induced stress.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

A) Representative pictures of human neuroblastoma BE-M17 cells transfected with WT, Q331K or Q343R TDP-43::EGFP. The Q331K and Q343R TDP-43::EGFP constructs produce more inclusions (arrows) than WT construct after treatment with arsenite (0.5 mM, 1 hr). However, co-treatment of arsenite and cycloheximide (CHX, 50 µg/ml, 1 hr) prevents formation of inclusions. B) Quantification of cytoplasmic inclusion formation for EGFP and TDP-43-EGFP constructs (WT, G294A, A315T, Q331K and Q343R) under different conditions (basal, arsenite and arsenite + cycloheximide). 15 fields were counted per condition. Scale bar  = 8 µm.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
4.
Figure 4

Figure 4. Relationship of TDP-43 to translation and cell death.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

A.) Representative immunoblot showing the effects of TDP-43 on protein translation. HEK-293 cells were transfected with GFP or TDP-43 (WT, TDP-4386–414 or TDP-43216–414). After 24 hrs they were treated ± arsenite (0–0.1 mM, 45 min), protein translation was assessed using click-chemistry labeling (15 min pulse). Arsenite treatment strongly inhibited translation of most proteins. TDP-43 expression was not associated with any consistent changes in translation of proteins. B.) Induction of caspase activity upon expression of TDP-43 constructs (WT = T-FL, TDP-43216–414 = T-216, TDP-4386–414 = T-86); control (Ctrl) refers to transfection with EGFP.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
5.
Figure 9

Figure 9. Disease-linked TDP-43 mutations are associated with increased levels of insoluble protein aggregates.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

HEK 293 cells were transfected with TDP-43 (WT, A315T or Q343R). After 48 hrs, the cells were treated ± arsenite (0.5 mM, 1 hr), separated into soluble and insoluble fractions, and immunoblotted. Upper panel: Soluble fraction. Lower panel: Insoluble fraction. Several different species of TDP-43 could be observed including bands putatively corresponding to endogenous TDP-43 (single star), TDP-43::EGFP (double star) and dimeric TDP-43::EGFP (triple star). Mutations were associated with increased levels of insoluble TDP-43 and increased levels of TDP-43 breakdown products (bands under the TDP-43::EGFP) after arsenite treatment.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
6.
Figure 10

Figure 10. Quantification of toxicity.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

A) Disease-linked mutations in TDP-43 increase caspase 3/7 activity after arsenite treatment. HEK 293 cells were transfected with EGFP, or TDP43 (WT, Q331K or Q343R). After 48 hrs the cells were treated ±50 µM arsenite, 12 hrs later caspase 3/7 activity was assayed under each condition. Results are shown normalized to EGFP transfected cells under basal conditions. B) BE-M17 cells were transfected with EGFP, or TDP43 (WT, G294A or Q331K). LDH release was measured after 48 hrs and normalized to total LDH and transfection efficiency. Baseline toxicity was set as the EGFP transfected neurons. Transfection with TDP-43 (WT, G294A or Q331K) increased LDH release, with G294A and Q331K TDP-43 causing significantly more release than WT TDP-43 (N = 6 measurements per group). C.) Induction of TDP-43 inclusions by arsenite (50 µM) at 12 hrs.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
7.
Figure 3

Figure 3. Co-localization of TDP-43 inclusions with SG markers.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

A.) Human neuroblastoma BE-M17 cells transfected with the TDP-4386–414 (T-86::GFP) construct form cytoplasmic inclusions under basal conditions (arrows). The inclusions co-localize with endogenous SG markers including, TIA-1, TIA-R and Poly A binding protein (PABP), identified by immunocytochemistry (ICC). B.) TDP-43 inclusions (WT = T-FL, TDP-43216–414 = T-216, TDP-4386–414 = T-86, green) occurring after arsenite treatment also co-localize with eIF3 (red). C.) Double-labeling of TDP-43 and Dcp1a (a P-body protein marker). BE-M17 cells were transfected with WT TDP-43::GFP = T-FL, TDP-4386–414::GFP = T-86 and TDP-43216–414 = T-216::GFP (green) and Dcp1a-mRFP (red). The Dcp1a::mRFP labels P-bodies (triangle), which are adjacent to TDP-43 positive inclusions (arrow). The boxed area is shown in higher magnification in the panels labeled “High Mag”. Scale bar  = 3 µm.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
8.
Figure 7

Figure 7. Regulation of TDP-43 inclusion formation by stress granule modulators.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

A) The translational inhibitors cycloheximide (50 µg/ml, 1 hr) or emetine (20 µg/ml, 1 hr) disperse inclusions formed by WT-TDP-43::EGFP (T-FL) TDP-4386–414::EGFP (T-86), while puromycin (20 µg/ml, 3 hrs) increases formation of TDP-4386–414::EGFP inclusions formation. Puromycin also induces formation of cytoplasmic inclusions formed by WT and TDP-4386–414::EGFP (arrows). However, puromycin does not change the abundance of TDP-43216–414::EGFP (T-216) inclusions. B) TDP-43::EGFP lacking the nuclear localization signal (TDP-43 K82/84A) forms abundant cytoplasmic inclusions that co-localize with TIA-1 (anti-TIA-1 antibody, red) after arsenite treatment. The inclusions are dispersed by co-treatment with cycloheximide (50 µg/ml, 1 hr). Quantification is shown in the bar graph to the right. C.) Cells were transfected with the TDP-43 ΔRRM1 and ΔRRM1/2 constructs (N-terminal EGFP tag). After 24 hrs the cells were subjected to two conditions: arsenite (0.5 mM, 1 hr) or arsenite plus cycloheximide (50 µg/ml, 1 hr). The cells were then fixed, and immunocytochemistry was performed TDP-43 inclusions co-localized with TIA-1. Arrows point to representative inclusions showing co-localization of TDP-43 and TIA-1. Cytoplasmic TDP-43 inclusion formation was not apparent under basal conditions, however under conditions of arsenite exposure both constructs formed inclusions that co-localized with TIA-1 and were cycloheximide reversible. Quantification is shown in the bar graphs to the right. Scale bar: 10 µm.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
9.
Figure 11

Figure 11. Co-localization of TDP-43 inclusions with SG markers (eIF3 and TIA-1) in ALS and FTLD-U brain.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

A) Treatment of human cortical brain sections with Sudan black strongly reduced endogenous fluorescence from lipofuscin in the red and green channels (subject 14, Table 1); Blue channel is DAPI staining. B) Co-localization of TIA-1 with TDP-43 in inclusions present in the spinal cord of a patient with ALS (Ctrl  =  subject 5, ALS  =  subject 1, Table 1). The boxed area is shown in higher magnification in the panels labeled “High Mag”. C) TDP-43 inclusions in the frontal cortex of a patient with FTLD-U co-localizes with eIF3 (Ctrl  =  subject 16, FTLD  =  subject 11, Table 1). FTLD-U and ALS sections with TDP-43 inclusions showed co-localization with SG markers. D) Co-localization of phospho-TDP-43 and eIF3 reactivity in FTLD-U brain (subject 10, Table 1). E) Controls for immunocytochemistry. Row 1: TDP-43 present in brain tissue from a patient without neurological disease shows predominantly nuclear localization (subject 14, Table 1). Row 2: TDP-43 positive cytoplasmic inclusion in a patient with FTLD-U showing co-localization with eIF3 (subject 9, Table 1). Rows 3 – 5: Demonstration of immunoabsorption with antigenic peptide, which eliminates staining by eIF3 (Control sample  =  neurological normal, subject 14; FTLD  =  subject 9, Table 1. Scale bars: A, B, C, D = 10 µm, E = 3 µm.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
10.
Figure 2

Figure 2. TDP-43 inclusions co-localize with SG proteins.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

A.) Cellular distribution of TDP-43::EGFP constructs in human neuroblastoma BE-M17 cells under basal conditions and after treatment with arsenite; the cells are co-labeled with anti-TIA-1 antibody (red, labeling SGs) and with DAPI (blue, labeling nuclei). Basal conditions (left panel): WT TDP-43 (T-FL) typically shows diffuse nuclear expression, while TDP-43216–414 (T-216) and TDP-4386–414 (T-86)::EGFP constructs form cytoplasmic and nuclear inclusions. TDP-43 reactivity co-localizes with RNA binding protein, TIA-1. Arsenite treatment (right panel): Treatment with arsenite (0.5 mM, 1 hr) induces some TDP-43::EGFP to translocate to the cytoplasm where it forms inclusions (arrows) that co-localize with TIA-1, indicating that the inclusions are SGs. The TDP-43216–414 and TDP-4386–414::EGFP constructs form inclusions in the cytoplasm (arrows) and nucleus, but only inclusions generated by the TDP-4386–414 construct are increased by arsenite. The inclusions present in arsenite treated cells co-localize with TIA-1. B.) Quantification of TDP-43 inclusions that co-localize with TDP-43 and TIA-1. C.) Incubation in Hanks balanced salt solution for 1 hr induced formation of TDP-43 inclusions (arrows) that co-localized with TIA-1 positive inclusions. Transfected EGFP did not form inclusions under the same conditions. Scale bar: 3 µm.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.
11.
Figure 6

Figure 6. Co-immunoprecipitation of TDP-43 and TIA-1.. From: Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue.

HEK 293 cells were transfected with TDP-43 (WT, K82/84A or A315T) and TIA-1. The K82/84A TDP-43 construct has had the nuclear localization signal removed (this construct is used again in figure 7B), and the A315T TDP-43 construct contains a mutation associated with familial ALS (this construct is used again in figure 8). The lysates were treated with RNase A (50 µg/ml) for 30 min at 37°C. Immunoprecipitated was performed with antibody to TIA-1, and the immunoblots were probed with antibody to TDP-43. Left upper panel: Abundant reactivity corresponding to the TDP-43::EGFP constructs(double star) was evident in lanes co-transfected TIA-1 with TDP-43::EGFP. A weaker band at 43 KD (single star) was also evident, suggesting association between TIA-1 and endogenous TDP-43. Finally, a higher molecular weight band, approximately consistent with the expected size of a TDP-43::EGFP dimer (∼140 KD) was also evident in the lane corresponding to A315T TDP-43 (triple star). Left lower panel: Following treatment with RNase A, binding to TDP-43 remained, although it was decreased (double stars). Treatment with RNase A abolished binding between TIA-1 and endogenous TDP-43 (single star), and decreased binding to the upper band (triple star), which presumably corresponds to the TDP-43::EGFP dimer. Right upper panel: Negative controls showing the specificity of the immunoprecipitation. Left lane: Lysate (30 µg) of transfected cells used for immunoprecipitation. Right lane: Immunoprecipitation using rabbit IgG instead of anti-TIA-1 antibody. The immunoblot was performed with anti-TDP-43 antibody.

Liqun Liu-Yesucevitz, et al. PLoS One. 2010;5(10):e13250.

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