Journal: Journal of Neuroscience

Article Title: Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

doi: 10.1523/jneurosci.6383-09.2010

Figure Lengend Snippet: Figure 1. Expression of WT and mutant GALC in mammalian cells. A–F, Empty pcDNA4 vector (Mock), human wild-type GALC cDNA(WT),orGLD-causingGALCmutations(I234T,L629R,andD528N)weretransientlytransfectedintoCOS-1cells(A–C)orstably transfected into H4 cells (D–F). GALC enzymatic activity, measured by in vitro colorimetric assay, was significantly reduced in lysates from cells transfected with each mutant GALC, compared with WT GALC (*p 0.05) (A, D). The precursor and processed forms of GALC were detected by Western blot using the CL1475 rabbit antibody against the N terminus of GALC (B, E), and the anti-V5 epitope tag antibody against the C terminus of GALC (C, F). Error bars indicate SEM.

Article Snippet: We purchased full-length, human, wild-type GALC cDNA from Origene.

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Transfection, Activity Assay, In Vitro, Colorimetric Assay, Western Blot

Journal: Journal of Neuroscience

Article Title: Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

doi: 10.1523/jneurosci.6383-09.2010

Figure Lengend Snippet: Figure 2. Analysis of the secretion and endocytosis of WT and mutant GALC. Condi- tioned medium was collected from H4 cells stably transfected with WT GALC, mutant GALC (I234T, L629R, and D528N), or empty vector (Mock), and was then applied to OLI-neu oligodendrocyte precursor cells for 24 h to determine the efficiency of GALC uptake. A, Western blot (anti-V5 epitope tag) showing detectable levels of GALC protein in condi- tioned media from WT, I234T, and D528N GALC-transfected H4 cells. Secreted GALC was undetectable in conditioned media from empty vector (mock)-transfected and L629R GALC-transfected cells. B, Western blot (CL1021AP chicken anti-GALC) of lysates from recipientOLI-neucellsafterincubationwithconditionedmediumfromH4cellsexpressing WT or mutant GALC. C, Summary of GALC activity determined by colorimetric assay from the H4 input media (A) and from the Oli-neu recipient lysates (B). Uptake index is defined as 100 times the ratio of the GALC activity measured in the cell lysate after uptake com- pared with the GALC activity in the corresponding input medium. The data in the table represent the mean values SE of triplicate samples in each group. GALC secretion and uptake were significantly decreased for each mutant compared with WT GALC (*p 0.05).

Article Snippet: We purchased full-length, human, wild-type GALC cDNA from Origene.

Techniques: Mutagenesis, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Colorimetric Assay

Journal: Journal of Neuroscience

Article Title: Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

doi: 10.1523/jneurosci.6383-09.2010

Figure Lengend Snippet: Figure 3. Confocal immunocytofluorescent analysis of GALC in H4-GALC cells. H4 cells (mock), WT GALC, and mutant (I234T, L629R and D528N) GALC-expressing H4 cells were seeded at subconfluentdensityonglasscoverslips2dbeforestaining.ThesubcellularlocalizationofGALCwasanalyzedbycostainingofGALC(CL1475rabbitanti-GALCantibody)andlamp2forlysosomes, costainingofGALC(anti-V5antibody)andcalnexinforER,andcostainingofGALC(anti-V5antibody)and58kGolgiproteinforGolgiapparatus.GALCandtheorganellemarkerswereillustratedin greenandredcolors,respectively.NucleiwerelabeledwithDAPIandillustratedinbluecolor.TheyellowcolorindicatescolocalizationofGALCwiththeorganellemarkers.Allimageswerecaptured in 40 magnification using a Zeiss LSM 510 META confocal microscope.

Article Snippet: We purchased full-length, human, wild-type GALC cDNA from Origene.

Techniques: Mutagenesis, Expressing, Microscopy

Journal: Journal of Neuroscience

Article Title: Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

doi: 10.1523/jneurosci.6383-09.2010

Figure Lengend Snippet: Figure 5. Western blot analysis of the hyperglycosylated D528N GALC mutant overex- pressed in COS-1 cells. A–C, “Polymeric” GALC (A), monomeric GALC precursors (B), and C-terminalGALCfragments(C)werepurifiedandanalyzedwithouttreatment(control)orafter deglycosylation by Endo H or by PNG F.

Article Snippet: We purchased full-length, human, wild-type GALC cDNA from Origene.

Techniques: Western Blot, Mutagenesis, Control

Journal: Journal of Neuroscience

Article Title: Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

doi: 10.1523/jneurosci.6383-09.2010

Figure Lengend Snippet: Figure 6. Expression study of D528N mutant GALC in the presence or absence of glycosyla- tionatposition528.PartialrescueinD528NGALCfunction,processing,secretion,endocytosis, andlysosomallocalizationwasobservedintheabsenceoftheglycosylationmotifthatisintro- duced with the D528N mutation. A, GALC activity in lysates from COS-1 cells transfected with emptyvector(mock),WTormutantGALC(D528N,S530AorD528NS530A)(top,graph).Data plottedrepresentthemeanSEofatleasttriplicatesamplesineachgroup.GALCactivitywas significantly increased in cells transfected with the double mutant D528NS530A, compared with the D528N mutant alone (*p 0.05). The same lysates were analyzed by Western blot using the CL1475 rabbit anti-GALC antibody (bottom, immunoblot). B, H4 cells stably trans- fected with WT and mutant GALC (D528N or D528NS530S) were used to determine the influenceofglycosylationonGALCsecretionanduptake.Conditionedmediumfromthetransfected

Article Snippet: We purchased full-length, human, wild-type GALC cDNA from Origene.

Techniques: Expressing, Mutagenesis, Activity Assay, Transfection, Western Blot, Stable Transfection

Journal: Journal of Neuroscience

Article Title: Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

doi: 10.1523/jneurosci.6383-09.2010

Figure Lengend Snippet: Figure 7. Activity and processing of GALC proteins in H4 cells under subphysiological tem- perature.WTandmutantGALC(D528N,I234T,L629R,andD528NS530A)transfectedH4cells wereculturedat37or30°Cfor72h.A,GALCenzymaticactivityinlysateswasdeterminedbyin vitro colorimetric assay. Data shown represent the mean SE of at least triplicate samples in eachgroup(*p 0.05).B,ProcessedN-terminalGALCfragments(50–54kDa)fromcelllysate were analyzed by Western blot using the chicken anti-GALC antibody, CL1021AP.

Article Snippet: We purchased full-length, human, wild-type GALC cDNA from Origene.

Techniques: Activity Assay, Colorimetric Assay, Western Blot

Journal: Journal of Neuroscience

Article Title: Molecular Characterization of Mutations That Cause Globoid Cell Leukodystrophy and Pharmacological Rescue Using Small Molecule Chemical Chaperones

doi: 10.1523/jneurosci.6383-09.2010

Figure Lengend Snippet: Figure 8. Effect of -lobeline on human GALC in vitro and in H4 cells expressing WT and mutantGALC(I234T,L629R,andD528N).A,TheinhibitionofrecombinantGALCby-lobeline wasanalyzedusinganinvitrocolorimetricenzymaticactivityassay.B,C,H4cellsexpressingWT GALC(B)orD528NGALC(C)weretreatedwithdifferentconcentrationsof-lobeline(60–240 M)for72h.GALCenzymaticactivityinthelysates(blackbars)andintheculturemedia(white bars)wasanalyzed.DatashownrepresentthemeanSEofatleasttriplicatesamplesineach group.Statisticalanalysiswasperformedbyone-wayANOVAtest,followedbytheTukey’spost hoc test (*p 0.05).

Article Snippet: We purchased full-length, human, wild-type GALC cDNA from Origene.

Techniques: In Vitro, Expressing

Journal: International Journal of Nanomedicine

Article Title: Combined Placental Mesenchymal Stem Cells with Guided Nanoparticles Effective Against Diabetic Nephropathy in Mouse Model

doi: 10.2147/IJN.S446733

Figure Lengend Snippet: Internalization of Nanoparticles (NPs) into Placental Mesenchymal Stem Cells (PL-MSCs). ( A ) MSCs stained with Prussian Blue to highlight iron (Fe) content. Scale bar = 50 µm. ( B ) Quantification of Fe concentration using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) (n=3). ( C ) Assessment of NP-induced cytotoxicity in MSCs labeled with NPs at varying concentrations for 24 h (n=4). ( D ) Measurement of β-galactosidase levels in NP-labeled MSCs using ELISA (n=4). ( E ) Proliferation rates of cells labeled with NPs at different concentrations (n=5). ( F and G ) Transmission Electron Microscopy (TEM) images showing NPs internalized into an MSC at a concentration of 50 µg/mL. The red arrow indicates NP. Data are presented as mean ± S.E.M. P -values < 0.05 were considered statistically significant, denoted as * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The supernatants were collected for β-galactosidase analysis using a human β-galactosidase ELISA kit (Molbio, Shanghai, China), in accordance with the manufacturer’s protocol.

Techniques: Staining, Concentration Assay, Clinical Proteomics, Spectroscopy, Labeling, Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy

Journal: ACS Omega

Article Title: Intracellular Delivery of β-Galactosidase Enzyme Using Arginase-Responsive Dextran Sulfate/Poly- l -arginine Capsule for Lysosomal Storage Disorder

doi: 10.1021/acsomega.7b01230

Figure Lengend Snippet: Cytotoxicity studies of bare DS/PA capsules with (a) R201C, (b) SV, (c) HeLa, and (d) wild-type mouse fibroblast cell lines.

Article Snippet: SV (β-galactosidase gene-deficient mouse fibroblast, JCRB1207) and R201C (deficient human β-galactosidase gene-introduced mouse fibroblast, JCRB1199), were purchased from the Japanese Collection of Research Bioresources cell bank.

Techniques: Capsules

Journal: ACS Omega

Article Title: Intracellular Delivery of β-Galactosidase Enzyme Using Arginase-Responsive Dextran Sulfate/Poly- l -arginine Capsule for Lysosomal Storage Disorder

doi: 10.1021/acsomega.7b01230

Figure Lengend Snippet: Uptake studies of DS/PA capsules with (a) SV, (b) R201C, (c) wild-type mouse fibroblast, and (d) HeLa cells after 12 h. (Note: Inset figures show Z-stack images of corresponding cells.)

Article Snippet: SV (β-galactosidase gene-deficient mouse fibroblast, JCRB1207) and R201C (deficient human β-galactosidase gene-introduced mouse fibroblast, JCRB1199), were purchased from the Japanese Collection of Research Bioresources cell bank.

Techniques: Capsules

Journal: ACS Omega

Article Title: Intracellular Delivery of β-Galactosidase Enzyme Using Arginase-Responsive Dextran Sulfate/Poly- l -arginine Capsule for Lysosomal Storage Disorder

doi: 10.1021/acsomega.7b01230

Figure Lengend Snippet: GM1 ganglioside levels in R201C cells (deficient human β-gal-gene-introduced mouse fibroblast) treated with free β-gal enzyme equivalent to the enzyme loaded in the capsules (top panel, a, c) and 50 β-gal-DS/PA capsules/cells (bottom panel, a) and 100 β-gal-DS/PA capsules/cells (bottom panel, c) at different time points (6, 12, and 24 h). FITC-cholera toxin B was used as a detection probe for GM1 ganglioside accumulation in culture fibroblasts. (b) and (d) represent the corresponding fluorescence intensity. Note: Control is untreated cells, and negative control is cells treated with bare DS/PA capsules (50 and 100 capsules/cell).

Article Snippet: SV (β-galactosidase gene-deficient mouse fibroblast, JCRB1207) and R201C (deficient human β-galactosidase gene-introduced mouse fibroblast, JCRB1199), were purchased from the Japanese Collection of Research Bioresources cell bank.

Techniques: Capsules, Fluorescence, Control, Negative Control

Journal: ACS Omega

Article Title: Intracellular Delivery of β-Galactosidase Enzyme Using Arginase-Responsive Dextran Sulfate/Poly- l -arginine Capsule for Lysosomal Storage Disorder

doi: 10.1021/acsomega.7b01230

Figure Lengend Snippet: GM1 ganglioside levels in treated (50 capsules/cell) and untreated R201C, SV, and wild-type mouse fibroblast cell line.

Article Snippet: SV (β-galactosidase gene-deficient mouse fibroblast, JCRB1207) and R201C (deficient human β-galactosidase gene-introduced mouse fibroblast, JCRB1199), were purchased from the Japanese Collection of Research Bioresources cell bank.

Techniques: Capsules