Journal: Nature neuroscience

Article Title: A glycolytic shift in Schwann cells supports injured axons

doi: 10.1038/s41593-020-0689-4

Figure Lengend Snippet: a, Top: Scheme of metabolomic analysis using extracts from nerve segments. Bottom: Concentrations of key energy metabolism intermediates in control and axotomized nerve segments from C57Bl/6J mice (F6P/G6P: fructose-6-phosphate/glucose-6-phosphate. FBP: fructose-1,6-bisphosphate. GI-OH3P: Glyceraldehyde-3-phosphate. 2PG/3PG: 2-phosphoglycerate/3-phosphoglycerate. LACT: lactate) (Error bars represent s.e.m. n=5 mice per condition and metabolite). b, Top: Scheme of extracellular flux analysis of SCs purified from C57Bl/6J mouse nerves. Bottom: Box and whiskers plot (maximum, 25th percentile, median, 75th percentile, minimum) shows glycolytic activity parameters as assessed by extracellular acidification rate (ECAR) measurements in control mouse SCs and cells with Nrg1-induced ErbB2 activation (n=9 well preparations per condition, *P=0.0080, **P=0.0464, ***P<0.0001). c, Western blot analysis (cropped blot images) of control- and Nrg1-treated C57Bl/6J mouse SCs probed with the indicated antibodies. (n=6 independent pair preparations (2 separate dishes) for PFKFB3, and n=3 independent pair preparations (2 separate dishes) for LDHA quantification). d, Intracellular and extracellular (supernatant) lactate concentrations from control and Nrg1-treated mouse SC preparations normalized to cell number and cellular protein. Note decreased intracellular and increased extracellular lactate levels in SCs treated with Nrg1 for 24h, indicating greatly increased lactate extrusion (Error bars represent s.e.m. n=3 well preparations from 3 independent experiments per condition).

Article Snippet: SCs were subsequently control-treated or treated with 200 ng/ml recombinant Nrg1 (R&D Systems, 396-HB-050) for 24h, collected in RIPA buffer containing phosphatase and protease inhibitors, and then processed for protein analysis and western blotting using standard procedures.

Techniques: Control, Purification, Activity Assay, Activation Assay, Western Blot

Journal: eLife

Article Title: A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction

doi: 10.7554/eLife.44530

Figure Lengend Snippet: ( A ) Western blot images of neuregulin 1-β1 (NRG1- β1; top) and β-actin (bottom) protein expression in three-independent ESC-derived MN cultures (N1 – N3). ( B ) Bar graph quantifying qRT-PCR results for epsilon subunit of AChR (CHRNE) gene expression in 2D (light blue) and 3D (blue bars) muscle alone, NRG1- β1 treated muscle cultures, and neuromuscular co-cultures. N = 3 muscle patient donors for data presented in ( B ). In ( B ) each symbol represents data from one muscle patient donor. Values in ( B ) are mean ±SEM. *p<0.05 and **p<0.01 compared to 3D muscle alone, and ## p<0.01 compared to 2D muscle culture. ( C ) Representative GCaMP6 epifluorescence images of 3D neuromuscular co-cultures pre-treated for 3 days with healthy (top) or myasthenia gravis (MG, bottom) patient IgG, together with human complement, and then stimulated with ACh (100 μM). Scale bars, 250 μm. ( D ) Bar graph indicating the percent total 3D tissue area occupied by muscle fibers responding to acetylcholine (ACh) stimulation in healthy and MG patient IgG treated neuromuscular co-cultures. n = 4 neuromuscular tissues treated with healthy IgG and three neuromuscular tissues each treated with serum IgG from one of three separate MG patient donors. Values in ( D ) are mean ±SEM. ***p<0.001.

Article Snippet: In experiments using neuregulin1-β1 treatment, recombinant human neuregulin1-β1 (R and D Systems) was supplemented in the culture media at 5 nM.

Techniques: Western Blot, Expressing, Derivative Assay, Quantitative RT-PCR

Journal: eLife

Article Title: A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction

doi: 10.7554/eLife.44530

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: In experiments using neuregulin1-β1 treatment, recombinant human neuregulin1-β1 (R and D Systems) was supplemented in the culture media at 5 nM.

Techniques:

Journal: Cell

Article Title: Inadequate DNA damage repair promotes mammary transdifferentiation leading to BRCA1 breast cancer

doi: 10.1016/j.cell.2019.06.002

Figure Lengend Snippet: KEY RESOURCES TABLE (antibodies and reagents)

Article Snippet: Sorted YFP + MECs were embedded in 15-20 μl Matrigel (Corning, 356231) in a 48-well plate, allowed to solidify for 5-10 mins, and then overlaid with DMEM/F12 advance media, supplemented with HEPES (1:100, GibcoTM, 15630080), GlutaMAX (1:100, GibcoTM, 35050-061), B27 (1:50, Thermo Fisher, 17504044), 100 ng/ml A83-01 (Tocris Bioscience, 2939), 50 ng/ml EGF (PeproTech, AF-100-15), 100 ng/ml Noggin (PeproTech, 120-10C), 500 ng/ml commercial R-spondin1 (Peprotech, 120-38) or conditioned R-spondin1 medium from RSPO cells (Cultrex, 3700-100-01), 100 ng/ml Nrg1 (R&D, 396-HB), 10 μM Y-27632 (ROCK inhibitor, Sigma Aldrich, Y0503) which is only necessary when organoids are first prepared, thawed, or when organoids are dissociated to single cells.

Techniques: Recombinant, Mutagenesis, Expressing, Software, Multiplex Assay