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: 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

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: The Journal of Biological Chemistry

Article Title: Phagocytosis-dependent ketogenesis in retinal pigment epithelium

doi: 10.1074/jbc.M116.770784

Figure Lengend Snippet: hfRPE generates β-HB through FAO of lipids from ingested outer segments. A, schematic representation of enzymatic reactions in ketogenesis. B, hfRPE expresses HMGCS2. C, β-HB is preferentially transported across the apical membrane of hfRPE after OS ingestion. hfRPE cells were incubated in apical chamber with glucose, OSs, or both, and the apical media were evaluated for β-HB content using a StanBio β-HB kit. *, p < 0.05; ***, p < 0.001., not significant. Plots are box-whisker plots showing median, with maximum and minimum range of the data for three independent experiments each done in triplicate. D, DHA serves as a substrate for FAO and ketogenesis, whereas oxidized OS and latex beads do not. hfRPE cells were incubated with glucose in the presence or absence of 200 μm DHA, 200 μm OS, 200 μm OxOS, or latex beads in apical chamber, and β-HB released was measured in the apical compartment using a StanBio β-HB kit. ***, p < 0.001. The values are means ± S.E. for three independent experiments, each done in triplicate.

Article Snippet: C , β-HB is preferentially transported across the apical membrane of hfRPE after OS ingestion. hfRPE cells were incubated in apical chamber with glucose, OSs, or both, and the apical media were evaluated for β-HB content using a StanBio β-HB kit.

Techniques: Incubation, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Phagocytosis-dependent ketogenesis in retinal pigment epithelium

doi: 10.1074/jbc.M116.770784

Figure Lengend Snippet: Generation of β-HB by ARPE19 cells is OS dose-dependent. A, ARPE19 cells express HMGCS2. B, β-HB is preferentially transported across the apical membrane of ARPE19 cells after OS ingestion. ARPE19 cells were incubated in apical chamber with glucose (Glc), OS, or OS plus glucose, and the apical supernatant was evaluated for β-HB content. **, p < 0.005. Plots are box-whisker plots showing median, with maximum and minimum range of the data for three independent experiments each done in triplicate. C, β-HB release increases as ingested OS levels increase. ARPE19 cells were incubated with glucose and increasing concentration of OSs (25–250 μm) in apical chamber for 3 h, and β-HB, the apical supernatant, was measured. *, p < 0.05; **, p < 0.005. The values are means ± S.E. for three independent experiments each done in triplicate. D, levels of opsin in ARPE19 cells challenged with increasing concentrations of OSs. Cleared ARPE19 cell lysates were prepared 3 h after OS challenge at the indicated concentrations and levels of opsin detected using anti-opsin 4D2 by immunoblot. E, ARPE19 cells were incubated with glucose and increasing concentration of OS-derived lipid liposomes in apical chamber for 3 h (25–200 μm), and then β-HB was measured in the apical compartment. **, statistical significance with p value < 0.005. The values are means ± S.E. for three independent experiments each done in triplicate. F, DHA serves as a substrate for FAO and ketogenesis, whereas oxidized OS and latex beads are not substrates. ARPE19 cells were incubated with glucose in the presence or absence of 200 μm DHA, 200 μm OS, 200 μm OxOS, or latex beads in the apical chamber, and β-HB was measured in the apical compartment content using a StanBio β-HB kit. ***, p < 0.001. The values are means ± S.E. for three independent experiments each done in triplicate.

Article Snippet: C , β-HB is preferentially transported across the apical membrane of hfRPE after OS ingestion. hfRPE cells were incubated in apical chamber with glucose, OSs, or both, and the apical media were evaluated for β-HB content using a StanBio β-HB kit.

Techniques: Incubation, Whisker Assay, Concentration Assay, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Phagocytosis-dependent ketogenesis in retinal pigment epithelium

doi: 10.1074/jbc.M116.770784

Figure Lengend Snippet: Defective phagosome maturation contributes to diminished apical β-HB. A, Western blot analysis of ARPE-19 cell lysates from cells stably transfected with control shRNA (C2) or MREG ShRNA (M5) both C2 and M5 cells have similar HMGCS2 levels. B, C2 and M5 ARPE-19 cells were incubated with glucose (5 mm) and OSs (200 μm) in the apical chamber for 3 h, and β-HB was measured in the apical compartment. Box-whisker plot representing the median and for three independent experiments each done in triplicate, with maximum and minimum range of the data. **, p < 0.005 compared with C2, with Student's t test.

Article Snippet: C , β-HB is preferentially transported across the apical membrane of hfRPE after OS ingestion. hfRPE cells were incubated in apical chamber with glucose, OSs, or both, and the apical media were evaluated for β-HB content using a StanBio β-HB kit.

Techniques: Western Blot, Stable Transfection, Transfection, shRNA, Incubation, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Phagocytosis-dependent ketogenesis in retinal pigment epithelium

doi: 10.1074/jbc.M116.770784

Figure Lengend Snippet: β-HB secretion from mouse RPE correlates with time of day. A, Hmgcs2 is preferentially expressed in the RPE. In situ hybridization with an Hmgcs2 specific probe (in red) using RNAscope. Confocal micrograph of albino retinal section stained for HMGCS2 probe using RNAscope technology (left) and transmitted light image to show the retinal layers (right). HMGCS2 transcript is highly restricted (red puncta) to the RPE. IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. B, localization of HMGCS2 to mitochondria in mouse RPE. Confocal micrograph of wild-type C57Bl6/J retinal sections immunostained for COXIV (green) and HMGCS2 (red) shows exclusively mitochondrial localization of HMGCS2 in the RPE. Nuclei are indicated in the merged image as blue. As expected, most of the staining is in the basolateral RPE. C, schematic representation of experimental design and RPE/choroid explant isolation. D, β-HB is released from mouse RPE/choroid explants upon OS ingestion. Supernatant was collected from RPE/choroid explants harvested as shown in C, and β-HB levels were determined immediately. *, p < 0.05; **, p < 0.005. Plots are box-whisker plots showing median, with maximum and minimum range of the data from six individual mice at each time point. E, HMGCS2 protein levels normalized to actin in RPE explants utilized for β-HB release experiments in D. Cleared lysates were isolated immediately after the 2-h sample was collected, and Western blotting for HMGCS2 was performed as described under “Experimental procedures.” **, p < 0.005. Plots are box-whisker plots showing median, with maximum and minimum range of the data for 12 eyes from 6 individual mice at each time point.

Article Snippet: C , β-HB is preferentially transported across the apical membrane of hfRPE after OS ingestion. hfRPE cells were incubated in apical chamber with glucose, OSs, or both, and the apical media were evaluated for β-HB content using a StanBio β-HB kit.

Techniques: In Situ Hybridization, Staining, Isolation, Whisker Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phagocytosis-dependent ketogenesis in retinal pigment epithelium

doi: 10.1074/jbc.M116.770784

Figure Lengend Snippet: β-HB secretion is delayed coincident with slowed phagosome maturation in mouse RPE. A, loss of MREG leads to phagosome accumulation in the RPE. Phagosome numbers per 100 μm of RPE length in Mreg+/+ and Mreg−/− mice at different time points relative to lights on. Counts were done in duplicate on 10 individual areas per mouse in a total of 2 mice/4 eyes with analyst blind to the strain of mouse. The results are means ± S.E. B, localization of HMGCS2 to mitochondria in mouse Mreg−/− RPE. Confocal micrographs of MREG−/− retinal sections immunostained for COXIV (green) and HMGCS2 (red) showing mitochondrial localization of HMGCS2 similar to WT. Nuclei are indicated in the merged image as blue. Note that the basolateral pattern of mitochondrial distribution closely resembling the WT. C, HMGCS2 protein levels in RPE explants utilized for β-HB release experiments in D. Cleared lysates were isolated immediately after the 2-h sample was collected, and Western blotting for HMGCS2 was performed as described under “Experimental procedures.” **, p < 0.005. Plots are box-whisker plots showing median, with maximum and minimum range of the data for 12 eyes from 6 individual mice at each time point. D, β-HB is released from mouse RPE/choroid explants upon OS ingestion. Supernatant was collected from RPE/choroid explants, and β-HB levels were determined immediately. *, p < 0.05; **, p < 0.005. the plots are box-whisker plots showing median, with maximum and minimum range of the data for 12 eyes from 6 individual mice at each time point.

Article Snippet: C , β-HB is preferentially transported across the apical membrane of hfRPE after OS ingestion. hfRPE cells were incubated in apical chamber with glucose, OSs, or both, and the apical media were evaluated for β-HB content using a StanBio β-HB kit.

Techniques: Isolation, Western Blot, Whisker Assay