α bungarotoxin fitc  (Alomone Labs)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TFEB safeguards trophoblast syncytialization in humans and mice.

doi: 10.1073/pnas.2404062121

Figure Lengend Snippet: Fig. 4. ERVFRD-1 is the direct target gene of TFEB responsible for governing STB formation. (A) Venn diagram depicting putative TFEB target genes by intersecting genes identified by TFEB ChIP-seq with down-regulated genes in TFEB KO BeWo cells. (B) Integrative Genomics Viewer track of TFEB binding near the predicted ERVFRD-1 promoter. (C) RT-qPCR analysis of ERVFRD-1 in TFEB KO cell lines with or without FSK treatment. n = 3. (D–H) Dual-luciferase reporter assays were conducted in HEK293T (D and F) or STBs (E,G, and H). WT (D and E) or truncated (F–H) ERVFRD-1 promoter reporter plasmids were introduced into the HEK293T or STBs. The promoter activities of ERVFRD-1 in HEK293T were evaluated in response to different dosages of TFEB overexpression (D, n = 3). The activities of ERVFRD-1 promoters in control, TFEB KO#1, and TFEB KO#2 STBs were assessed after 72 h differentiation (E, n = 3). Similarly, the WT or mutant (with single or double TFEB binding sites deletion) ERVFRD-1 promoter activities in HEK293T (F, n = 3), STBs (G, n = 5), or GCM1 KO STBs (H, n = 3) were determined. (I) Immunofluorescence images of CDH1 in TFEB KO hTSCs upon reexpressing exogenous SYNCYTIN2. Vector control, empty pCDH-EGFP plasmid. Overexpression efficiency was validated by the EGFP immunofluorescence in the corresponding lower panels.

Article Snippet: Intriguingly, we observed the enrichment of TFEB binding in the promoter region of the ERVFRD- 1 gene (Fig. 4B), a well- characterized fusogen facilitating human Down regulated genes in KO vs WT TFEB ChIP-seq 38 A B E F D G H 4000 100 200 300 GCM1 KO Control TFEB KO#1 TFEB KO#2 Vector control SYNCYTIN2 OE C D H 1 EG FP C luc luc luc pcDNA3.1TFEB pcDNA3.1 Predicted TFEB-Binding Site luc luc 0 2 4 6 8 10 100 200 300 Promoter activity of ERVFRD-1(a.u) -600bp TSS-0 pcDNA3.1 12.5 25 50 100 0 200 400 600 800 1000 pcDNA3.1-TFEB(ng) Lu ci fe ra se a ct iv ity of E R VF R D -1 (a .u ) Co ntr ol TF EB KO #1 TF EB KO #2 0 100 200 300 400 Pr om ot er ac tiv ity of E R VF R D -1 (a .u ) STBs Culture Condition 0 100 200 300 400 500 Promoter activity of ERVFRD-1(a.u) STBs Culture Condition luc luc luc luc STBs Culture ConditionSTBs Culture Condition 0 5 10 15 20 25 100 150 200 Re la tiv e ER VF RD -1 ex pr es sio n 293T Co ntr ol Co ntr ol TF EB KO #1 TF EB KO #1 TF EB KO #2 TF EB KO #2 DMSO FSK I luc luc luc luc luc 11,100 kb 11,102 kb 11,104 kb 11,106 kb 11,108 kb 11,110 kb 11,112 kb 11,114 kb [0 - 31] Chr6: 11,097,771-11,115,165 TFEB-IP Input ERVFRD-1 CLEAR Motif CLEAR Motif CLEAR Motif 20μm SYNCYTIN2 OEVector control 370 (408) 1039 (1077) p=0.0175 p=0.0127 p<0.0001 p<0.0001 p=0.0199 p=0.0005 p<0.0001 p<0.0001 p<0.0001 p<0.0001 p=0.0002 p<0.0001 p=0.0002 p<0.0001 p=0.0001 p<0.0001 p<0.0001 -600bp TSS-0 -600bp TSS-0 Promoter activity of ERVFRD-1(a.u) Fig. 4.

Techniques: ChIP-sequencing, Binding Assay, Quantitative RT-PCR, Luciferase, Over Expression, Control, Mutagenesis, Immunofluorescence, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1 G93A Model

doi: 10.1038/s41419-019-1451-2

Figure Lengend Snippet: a Representative images of H&E-stained cross-sections from Gastrocnemius muscle of mice from 4 groups: WT-vehicle, WT-30mpk pridopidine, SOD1 G93A -vehicle, and SOD1 G93A -30mpk pridopidine. The muscle histology of SOD1 G93A -vehicle mice is poor and reveals a smaller diameter of muscle fiber, compared with WT-vehicle and WT-30mpk pridopidine. Muscles from pridopidine-treated SOD1 G93A mice exhibit a larger and more homogenous muscle fiber population that resembles WT muscles. b Representative confocal images of muscle whole-mount preparations from 16-week-old mice stained for Postsynaptic AchR (BTX; red) and Presynaptic NFH + Synapsin-I + Synaptophysin (green). Muscles of SOD1 G93A vehicle-treated mice show the degeneration of presynaptic neurons and the amorphous post-synaptic apparatus. SOD1 G93A -30mpk pridopidine-treated mice display co-localized pre- and post-synaptic markers with a typical healthy NMJ morphology. c Quantitative analysis of muscle fiber diameter indicates that SOD1 G93A -vehicle-treated muscle fibers are significantly smaller in diameter than are WT-vehicle fibers. 30mpk of pridopidine led to a significant ~4 µm increase in the muscle fiber diameter in SOD1 G93A and ~5 µm in WT muscles. (n = number of muscle fibers.) d Quantitative analysis of the percentage of innervated NMJs reveals a massive ~60% loss of NMJs in the SOD1 G93A -vehicle group, compared with the WT-vehicle group. Pridopidine treatment limited the loss of NMJs in SOD1 G93A mice to ~20%. Data are shown as mean ± SEM ( n = number of NMJs). * p value < 0.05; ** p value < 0.01; *** p value < 0.001 ( n = 5 mice in each group; Student’s t test)

Article Snippet: At 7 days of co-culture, cultures were fixed and the myocytes in the NMJ compartment were stained with α-Bungarotoxin-FITC 1:30 (Alamone Labs; B-100-F) to label the extracellular domain of postsynaptic nAchR.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1 G93A Model

doi: 10.1038/s41419-019-1451-2

Figure Lengend Snippet: a Schematic illustration of the experimental procedure. Primary skeletal myoblasts from presymptomatic (P60) WT or SOD1 G93A mice are cultured in the distal compartment of a microfluidic chamber. Seven days later, ventral spinal cord explants from WT or SOD1 G93A E12.5 mouse embryos are plated in the proximal compartment. After 7 more days, axons extend into the distal compartment and generate connections and NMJs with differentiated myocytes. b High-magnification fluorescent images of differentiated myotubes in the NMJ compartment labeled for nuclei (blue) and the Ryanodine Receptors (RyR, yellow). Images demonstrate that WT (left) and SOD1 G93A (right) myocytes differentiate to the same extent, as can be seen by the peripheral localization of the nuclei, and by the striated organization of RyR. c Upper panel: Phase image of a myocyte in the distal compartment connected by axons (green arrowheads). Scale bar: 20 µm. Lower panel: High magnification images of myocyte:MN contact points reveals the formation of NMJs as seen by co-localization of post-synaptic AChRs (red) with HB9::GFP axons (green) and 3-dimensional colocalization of pre- and post-synaptic markers (Cyan). The image below the inset shows an orthogonal slice displaying Z-axis colocalization. Scale bars: 10 µm; inset 5 µm. d Muscle contraction traces as extracted from intensity over time measurements of muscle contraction show the flat trace of a non-contracting, immobile myocyte (upper), and the trace of a contracting myocyte demonstrating multiple bursting events (lower). e Bar chart of the percentage of contracting myofibers in co-culture shows that only ~10% of muscles contract in the absence of MNs versus 74% in healthy co-cultures. Co-culture combinations that include at least one of the cell types expressing SOD1 show a significantly lower percentage of contracting myocytes. (n = number of microfluidic chambers from 3 independent experiments). f Bar chart of the percentage of contracting myocytes in co-culture show that the addition of pridopidine to the culture medium of co-cultures with SOD1 G93A myocytes (0.1 and 1 µM) restores neuromuscular activity to the WT levels. Combination of S1R −/− MNs results in even a lower number of contracting myotubes. Application of 0.1 µM pridopidine to S1R − / − co-cultures does not restore the neuromuscular activity, as seen for the same concentration of pridopidine in co-cultures with WT neurons. Application of 1 µM pridopidine leads to an increase in the percentage of contracting myocytes that is significantly lower than untreated and treated WT cultures. g Representative images of in-vitro NMJs in the distal compartment display co-localized presynaptic motor axons (red) and AchR patches (green) on myocytes (circled by white dashed lines). The left panel shows that the co-localized axons in the WT co-cultures are intact, whereas axons in SOD1 G93A co-cultures are degenerated (center panel). SOD1 G93A co-cultures treated with 0.1 µM pridopidine display healthy NMJs and a reduced number of degenerated axons (right panel). Scale bar: 40 µm. h Quantitative analysis of in vitro NMJs reveals a profound reduction in the percentage of healthy NMJs in SOD1 G93A co-cultures that is significantly moderated by 0.1 µM pridopidine treatment. Data are shown as mean ± SEM. * p value < 0.05; ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001. ( n = number of microfluidic chambers from 3 or more independent experiments; Student’s t test)

Article Snippet: At 7 days of co-culture, cultures were fixed and the myocytes in the NMJ compartment were stained with α-Bungarotoxin-FITC 1:30 (Alamone Labs; B-100-F) to label the extracellular domain of postsynaptic nAchR.

Techniques: Cell Culture, Labeling, Co-Culture Assay, Expressing, Activity Assay, Concentration Assay, In Vitro

Journal: Cell Death & Disease

Article Title: Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1 G93A Model

doi: 10.1038/s41419-019-1451-2

Figure Lengend Snippet: a Representative images of H&E-stained cross-sections from Gastrocnemius muscle of mice from 4 groups: WT-vehicle, WT-30mpk pridopidine, SOD1 G93A -vehicle, and SOD1 G93A -30mpk pridopidine. The muscle histology of SOD1 G93A -vehicle mice is poor and reveals a smaller diameter of muscle fiber, compared with WT-vehicle and WT-30mpk pridopidine. Muscles from pridopidine-treated SOD1 G93A mice exhibit a larger and more homogenous muscle fiber population that resembles WT muscles. b Representative confocal images of muscle whole-mount preparations from 16-week-old mice stained for Postsynaptic AchR (BTX; red) and Presynaptic NFH + Synapsin-I + Synaptophysin (green). Muscles of SOD1 G93A vehicle-treated mice show the degeneration of presynaptic neurons and the amorphous post-synaptic apparatus. SOD1 G93A -30mpk pridopidine-treated mice display co-localized pre- and post-synaptic markers with a typical healthy NMJ morphology. c Quantitative analysis of muscle fiber diameter indicates that SOD1 G93A -vehicle-treated muscle fibers are significantly smaller in diameter than are WT-vehicle fibers. 30mpk of pridopidine led to a significant ~4 µm increase in the muscle fiber diameter in SOD1 G93A and ~5 µm in WT muscles. (n = number of muscle fibers.) d Quantitative analysis of the percentage of innervated NMJs reveals a massive ~60% loss of NMJs in the SOD1 G93A -vehicle group, compared with the WT-vehicle group. Pridopidine treatment limited the loss of NMJs in SOD1 G93A mice to ~20%. Data are shown as mean ± SEM ( n = number of NMJs). * p value < 0.05; ** p value < 0.01; *** p value < 0.001 ( n = 5 mice in each group; Student’s t test)

Article Snippet: At 7 days of co-culture, cultures were fixed and the myocytes in the NMJ compartment were stained with α-Bungarotoxin-FITC 1:30 (Alamone Labs; B-100-F) to label the extracellular domain of postsynaptic nAchR.

Techniques: Staining