Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) Cdc48-GFP strain alone (left), and Cdc48-GFP strain transformed with a TDP-43-mRuby2 plasmid (right) were imaged in both mid log (M-L) and stationary phase (S-P). Arrowhead and percentage values indicate Cdc48 and TDP-43 co-localization. Scale bar: 2 µm. (B) Cdc48-TAP strain was transformed with an empty vector or TDP-43-YFP plasmid, and then TAP immunoprecipitation was performed. (C) As in (B), but with GFP immunoprecipitation i.e. reciprocal pull down. (D) Cdc48-TAP strain with TDP-43 was transformed with vector or Pab1-GFP and the interaction was determined. (E) WT and cdc48-3 strains were treated with 0.2 mg/ml Cycloheximide (CHX) for the indicated time at either 25°C or 35°C. TDP-43 protein levels detected and normalized relative to a PGK1 loading control. (F) Serial dilution growth assay of WT and cdc48-3 transformed with empty vector or GAL1 -regulated TDP-43-YFP plasmid at different temperatures.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Transformation Assay, Plasmid Preparation, Immunoprecipitation, Serial Dilution, Growth Assay

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) Growth assay of WT and indicated isogenic null strains transformed with an empty vector or GAL1 -regulated TDP-43-GFP plasmid. (B) Ubx3-GFP strain was transformed with a TDP-43-mRuby2 plasmid and images were taken at mid-log phase. Arrowhead indicates Ubx3 and TDP-43 co-localization. Scale bar: 2 µm. (C) Serial dilution growth assay of indicated strains transformed with GAL1 -regulated TDP-YFP plasmid (D) Cdc48-GFP strain was transformed with an untagged TDP-43 plasmid and either a Vps34-or Vps38-mRuby2 plasmid; Arrowhead indicates Cdc48 co-localization with both Vps34 and Vps38. Scale bar: 2 µm.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Growth Assay, Transformation Assay, Plasmid Preparation, Serial Dilution

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) WT and cdc48-3 cells were cultured at 35°C for 2 hours and then stained with FM4-64 dye (8 uM) for time periods indicated and examined. Arrowhead indicates vacuolar staining. Persistence of plasma membrane staining in cdc48-3 cells and reduced vacuolar membrane staining relative to WT indicates endocytic defect. (B-C) As in (A) except with additional expression of TDP-43-GFP (B) or FUS-GFP (C). Scale bar = 2 µm. (D) Analysis of vacuolar staining intensity of (A-C). **P < 0.01, ***P < 0.001 by Student’s unpaired two-tailed t test. Data was shown as mean ± s.e.m.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Cell Culture, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: (A) HEK 293A cells were either treated with 40µM dynasore for 24 hours or subject to VCP knock down, followed by determination of TDP-43 and FUS protein levels and analyzation. (B) HEK293A cells expressing stably integrated TDP-43-GFP or TDP-35 YFP were examined under normal growth or following DBeQ treatment (10µM, 1hr). Scale bar: 5µm (C-D) Fluorescence immunohistochemistry of control (CTL) and ALS patients (P) frontal cortex tissue with TDP-43, VCP and DAPI and quantification. Arrowhead indicates VCP co-localization with TDP-43. Scale bar: 5 µm. *P < 0.05 by Student’s unpaired to-tailed t test. Data is shown as mean ± s.e.m.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Expressing, Stable Transfection, Fluorescence, Immunohistochemistry

Journal: bioRxiv

Article Title: Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

doi: 10.1101/668798

Figure Lengend Snippet: In yeast, the toxicity, turnover and aggregation of both TDP-43 and FUS depend on endocytosis function (this study and ). We suggest that large microscopically visible aggregates of TDP-43 and FUS are most likely cleared from the cytoplasm by autophagy, with oligomeric or soluble forms of TDP-43 and FUS being subject to clearance by endocytic or proteasomal means. If true, given our yeast toxicity data, in which endocytic but not autophagic mutants enhance TDP-43 and FUS toxicity, this would suggest that oligomeric, rather than microscopically visible forms of TDP-43 and FUS are the “toxic” cellular species. Cdc48/VCP, which localizes in microscopically visible TDP-43 or FUS aggregates, may facilitate TDP-43 and FUS conversion into oligomeric and soluble forms. Red arrows broadly represent antagonistic aggregation-promoting processes which may include cellular stress, impaired proteostasis, RNA metabolism defects and perturbation of nuclear-cytoplasmic trafficking, all of which are implicated in ALS pathology. Cdc48/VCP may facilitate entry of TDP-43 and FUS into the endocytic pathway. Finally, sequestration of Cdc48/VCP (and various endocytic proteins) within TDP-43 or FUS aggregates may contribute to the observed inhibition of endocytosis rates caused by TDP-43 or FUS expression, owing to various roles described for Cdc48/VCP in the endocytic pathway. The means by which TDP-43 and FUS enter the endocytic pathway remains mechanistically undefined.

Article Snippet: Primary antibodies were as follows: α-GFP (902602; Biolegend), α-TDP-43 (60019-2-Ig; ProteinTech), α-Vinculin (V4505; Sigma-Aldrich), α-Pgk1 (ab113687; Abcam), α-VCP (ab11433; Abcam), α-TAP (Thermofisher CAB1001) and α-GAPDH (MA5-15738, Invitrogen).

Techniques: Inhibition, Expressing

Journal: PLoS ONE

Article Title: Demineralized bone matrix used for direct pulp capping in rats

doi: 10.1371/journal.pone.0172693

Figure Lengend Snippet: (A): Representative immunohistochemical images of the blank control group, the Ca(OH) 2 group and the DBM group. a: The blank control group. Pulp morphology was normal. b- f: Ca(OH) 2 group (1, 3, 7, 14, 28 days). g- k: DBM group (1, 3, 7, 14, 28 days). (B): Mean IOD value of Runx2. *means significant difference.

Article Snippet: The sections were deparaffinized with xylene, hydrated in a series of descending grades of ethanol, and then rinsed briefly with PBS for the primary antibodies; the sections were incubated overnight at 4°C with polyclonal anti- Runx2, COL I, OCN and DSP (Wuhan Boster Biological Technology, Wuhan, China).

Techniques: Immunohistochemical staining, Control

Journal: bioRxiv

Article Title: Circulatory proteins shape microglia state and boost phagocytosis

doi: 10.1101/2024.09.30.615861

Figure Lengend Snippet: Representative flow cytometry data of pre-gated microglia (CD45 low CD11b + CD206 - ) in the hypothalamus region of NHS-Atto647N labeled individual protein-injected mouse comparing to a saline-injected mouse (3-month-old). The percentages of protein-positive microglia are shown. A) Experimental scheme for testing the uptake of NHS-Atto647N labeled individual protein in microglia by flow cytometry. B) Mice injected with Clusterin or Fibrinogen are compared to the saline-injected group. C) Mice injected with labeled ApoM, ApoE or Albumin are compared to the saline-injected group. D) Mice injected with Vitronectin or Fibronectin are compared to the saline-injected group. E) Mice injected with Plasminogen or Vitamin D-binding protein (DBP) are compared to the saline-injected group. F) Mouse injected with Transferrin is compared to the saline-injected group. G) Mouse injected with ApoA-II is compared to the saline-injected group.

Article Snippet: ApoA-I protein (Athens Research &Technology, 16-16-120101-LEL), ApoA-II (Athens Research & Technology, 16-16-120102), ApoM (LSbio, LS-G14868), ApoE (Sigma, SRP4760), Albumin (Sigma, 126674), Clusterin (Sino Biological, 50485), Fibrinogen (Abcam, ab92791), Vitronectin (Sino Biological, 50585-M08H), Fibronectin (Corning, 354008), Plasminogen (Innovative research, IMSPLG1MG), Vitamin D-binding protein (DBP) (Athens Research &Technology, 16-16-070307), and Transferrin (Sigma, T0665) were used for labeling.

Techniques: Flow Cytometry, Labeling, Injection, Saline, Binding Assay