Journal: Journal of neuroscience research

Article Title: Polyomavirus JC Infection Inhibits Differentiation of Oligodendrocyte Progenitor Cells

doi: 10.1002/jnr.23135

Figure Lengend Snippet: Preparation of human neural progenitor cells (hNPC) and their differentiation into human oligodendrocyte progenitor cells (hOPC) and human oligodendrocytes (hOL). A: Schematic representation of the preparation of hNPC from human fetal brain tissue (hfB, embryonic age 16 weeks) based on protocols of Espinosa-Jeffrey at al. (2009), with slight modifications. After dissociation of brain tissue, cells were plated onto nontissue-culture-grade Petri dishes coated with anti-PSA-NCAM antibody. Unattached cells were cultured in neural stem cell medium (NSCM) either as two- or as three-dimensional hNPC cultures. The hNPC were then gradually switched mixed with glial defined medium plus growth factors (GDM+) to differentiate them into hOPC and then to glial defined medium minus growth factors (GDM) to differentiate them into hOL. B: The hNPC stage of cultures was ascertained by immunolabeling of hNPC plated on slides with antibodies specific to nestin, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. C: Immunolabeling of hOPC for expression of nestin, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. D: Immunolabeling of hOL cultures for expression of GalC, A2B5, βIII-tubulin, and GFAP. Nuclei were stained with DAPI. Average percentages of cells expressing specific markers were quantified in at least 15 fields of view (~700–800 cells total). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: After removal of meninges and dissociation of brain tissue in the presence of Tryple Express (Gibco, Grand Island, NY; catalog No. 12605) and DNase I (10 U/ml; Sigma, St. Louis, MO), cells were triturated through a fire-polished Pasteur pipette and plated onto nontissue-culture-grade Petri dishes coated with anti-PSA-NCAM antibody (Developmental Studies Hybridoma Bank, University of Iowa [DSHB]; 5A5) in neural stem cell medium (NSCM: Neurobasal medium; Invitrogen, Carlsbad, CA; catalog No. 10888-022), N2 supplement (Invitrogen; catalog No. 17502048), B27 supplement without vitamin A (Invitrogen; catalog No. 12587-010), GlutaMAX (Invitrogen), 10 ng/ml basic fibroblast growth factor (bFGF; Invitrogen), 10 ng/ml recombinant human epidermal growth factor (EGF; Invitrogen), penicillin/streptomycin, and fungizone.

Techniques: Cell Culture, Immunolabeling, Staining, Expressing

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet:

Article Snippet: Library quantity, purity and size selection were assessed using Qubit fluorometer (Qubit DNA Assay kit, Invitrogen Q32854) and an Agilent Bioanalyzer (D5000 ScreenTape, Agilent 5067–5592) with acceptable library size distribution between 600 and 1150bp.

Techniques: Recombinant, Antibody Labeling, Staining, Cell Isolation, Control, Mass Cytometry, Sequencing, Software

Journal: Cell

Article Title: Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease

doi: 10.1016/j.cell.2018.08.067

Figure Lengend Snippet:

Article Snippet: Anti-Human CD326/EpCAM (9C4)-141Pr , Fluidigm , Cat#3141006B; RRID: AB_2687653.

Techniques: Recombinant, Coagulation, Antibody Labeling, Membrane, Plasmid Preparation, Polymer, Blocking Assay, Staining, Imaging, cDNA Synthesis, RNAscope, DNA Library Preparation, Software, Hybridization

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 1. miR‑214 is downregulated in liver cancer and targets Wnt3a. (A) Reverse transcription‑quantitative polymerase chain reaction was performed to examine the expression of miR‑214 in 24 paired human hepatocellular carcinoma and non‑tumor tissues. (B) Relative expression of miR‑214 in liver cancer cell lines and a normal liver cell line. **P<0.01; *P<0.05. (C) miR‑214 seed region sequence in the 3'UTR of Wnt3a. (D) Wnt3a protein expression as detected by immunohistochemistry. (E) Protein expression levels of Wnt3a were measured by western blot analysis in HepG2 cells transfected with miR‑214 or miR‑ctrl. (F) miR‑214 was co‑transfected with pmirGLO, pmirGLO‑Wnt3a‑3'‑UTR‑wt or pmirGLO‑Wnt3a‑3'‑UTR‑mut in HepG2 cells. Relative luciferase activity was measured after 48 h. *P<0.05 vs. control. miR, microRNA; mut/M, mutant; UTR, untranslated region; wt/W, wild‑type.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Polymerase Chain Reaction, Expressing, Sequencing, Immunohistochemistry, Western Blot, Transfection, Luciferase, Activity Assay, Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 2. miR‑214 inhibits the proliferation of liver cancer cells. CCK8 assay was performed to detect the effects of miR‑214 on cell proliferation at 24, 48, and 72 h in (A) HepG2 and (B) Hep3B cells. CCK8 assay was performed to detect the effects of siWnt3a on cell proliferation at 24, 48 and 72 h in (C) HepG2 and (D) Hep3B cells. Wnt3a overexpression vector was co‑transfected with miR‑ctrl or miR‑214 into (E) HepG2 and (F) Hep3B cells, and cell proliferation was detected by CCK8 assay. *P<0.05; **P<0.01 vs. miR‑ctrl + Wnt3a‑ctrl. CCK8, Cell Counting kit‑8; ctrl, control; miR, microRNA; OD, optical density; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: CCK-8 Assay, Over Expression, Plasmid Preparation, Control, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 3. Overexpression of miR‑214 or Wnt3a silencing affects cell cycle progression. Cell cycle analysis of (A) HepG2 and (B) Hep3B cells following transfection with miR‑214 or miR‑ctrl for 48 h. Cell cycle analysis of (C) HepG2 and (D) Hep3B cells following transfection with siWnt3a or si‑ctrl for 48 h. *P<0.05. ctrl, control; miR, microRNA; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Over Expression, Cell Cycle Assay, Transfection, Control, Small Interfering RNA

Journal: The Journal of Biological Chemistry

Article Title: An Isozyme-specific Redox Switch in Human Brain Glycogen Phosphorylase Modulates Its Allosteric Activation by AMP * ^♦

doi: 10.1074/jbc.M116.757062

Figure Lengend Snippet: Human bGP is inhibited by H2O2 through modification of cysteine residues. a, purified recombinant human bGP and lGP (white and gray bars) or purified muscle glycogen phosphorylase (mGP) (rabbit) (black bars) were incubated with or without 250 μm H2O2 for 30 min at 37 °C, prior to residual activity measurement. mGP and bGP activity was measured using AMP as an activator, whereas lGP, which only responds to phosphorylation, was phosphorylated by phosphorylase kinase for activation prior to activity measurement. Results are expressed as the percentage of the control without H2O2. Data represent mean values of three independent experiments ± S.D., ***, p < 0.001 when compared with control (no H2O2). b, reduced recombinant bGP was incubated with different concentrations of H2O2 (0–250 μm) for 30 min at 37 °C. Residual activity was assayed, and aliquots were also subjected to Western blotting analysis under non-reducing conditions. bGP was revealed using specific antibodies. Results are expressed as the percentage of the control. Data represent mean values of three independent experiments ± S.D., ***, p < 0.001 when compared with positive control. c, reduced recombinant bGP was incubated with glucose oxidase (1.2 units) and glucose (5 mm) for 30 min. In these conditions, H2O2 was continuously generated at a rate of 6 μm/min. An aliquot was removed every 5 min and assayed for bGP residual activity (filled circles). A control reaction was carried out in the presence of 300 units/ml catalase (open circles). Glucose, glucose oxidase, and catalase, independently, had no effect on bGP activity. Samples were analyzed by Western blotting, and bGP was revealed using specific antibodies. Results are expressed as the percentage of the control. Data represent mean values of three independent experiments ± S.D., ***, p < 0.001 when compared with t0. d, to confirm the oxidation of cysteine residues upon exposure to H2O2, recombinant bGP was inhibited by H2O2, and free cysteine residues were specifically labeled using the fluorescent probe 5-IAF. Samples were run on SDS-PAGE in the presence of 2-mercaptoethanol and blotted onto nitrocellulose membrane. 5-IAF fluorescence was measured (λex: 492 nm; λem: 520 nm), and bGP was revealed using specific antibodies. The untreated bGP was used as a positive control. To assess the number of oxidized cysteines after exposure to H2O2, thiol content was analyzed using the DTNB assay on both treated and untreated bGPs, as described under “Experimental Procedures.” The resulting TNB− formation was quantified by absorbance measurement at 405 nm. Absorbance of the non-treated protein was used as control. Data represent mean values of three independent experiments ± S.D., **, p < 0.01 when compared with reduced control. e, recombinant bGP was incubated with various concentrations of H2O2 (0–400 μm H2O2). Aliquots were removed every 5 min and assayed for residual activity. For each concentration of H2O2, the plot of the natural logarithm as a function of time allowed the determination of first-order apparent constants kobs. f, the second-order rate constant was then determined by plotting the first-order apparent constants against [H2O2] (lower panel). The solid lines represent the best linear regression fit of the data to Equations 2 and 3. The calculated kinact for the inhibition of bGP by H2O2 was 185 m−1 min−1. Data represent mean values of three independent experiments ± S.D.

Article Snippet: The oligonucleotides used for the site-directed mutagenesis were purchased from Eurofins. pCMV6 carrying bGP cDNA (pCMV6- PYGB ) was purchased from OriGene Technologies, Inc. Antibodies raised against brain glycogen phosphorylase were obtained from Santa Cruz Biotechnology (SC-81751).

Techniques: Modification, Purification, Recombinant, Incubation, Activity Assay, Phospho-proteomics, Activation Assay, Control, Western Blot, Positive Control, Generated, Labeling, SDS Page, Membrane, Fluorescence, DTNB Assay, Concentration Assay, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: An Isozyme-specific Redox Switch in Human Brain Glycogen Phosphorylase Modulates Its Allosteric Activation by AMP * ^♦

doi: 10.1074/jbc.M116.757062

Figure Lengend Snippet: bGP is inhibited in cells exposed to H2O2. a, HEK293T cells were transfected or not transfected with pCMV6-PYGB plasmid, and cells were exposed to 500 μm H2O2 for 20 min before being harvested. Cell lysis was then performed in the presence or absence of a reducing agent (10 mm DTT), and whole-cell extracts were assayed for endogenous glycogen phosphorylase activity. Data are expressed as the percentage of the control and represent mean values of three independent experiments ± S.D. ***, p < 0.001 when compared with positive control (upper panel); ###, p < 0.001 when two non-control groups are compared. Non-reduced and reduced whole-cell extracts were Western blotted and revealed for brain glycogen phosphorylase using an anti-bGP antibody. Ponceau red stains of the membranes are shown (lower panel). b, U87MG cells were exposed to 500 μm H2O2 for 20 min before being harvested. Cell lysis was then performed in the presence or absence of a reducing agent (10 mm DTT), and whole-cell extracts were assayed for endogenous glycogen phosphorylase activity. Data are expressed as the percentage of the control and represent mean values of three independent experiments ± S.D. ***, p < 0.001 when compared with positive control (upper panel); ###, p < 0.001 when two non-control groups are compared. Western blotting analysis of bGP from cells was revealed for brain glycogen phosphorylase using anti-bGP antibodies. Ponceau red stains of the membranes are shown (lower panel).

Article Snippet: The oligonucleotides used for the site-directed mutagenesis were purchased from Eurofins. pCMV6 carrying bGP cDNA (pCMV6- PYGB ) was purchased from OriGene Technologies, Inc. Antibodies raised against brain glycogen phosphorylase were obtained from Santa Cruz Biotechnology (SC-81751).

Techniques: Transfection, Plasmid Preparation, Lysis, Activity Assay, Control, Positive Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: An Isozyme-specific Redox Switch in Human Brain Glycogen Phosphorylase Modulates Its Allosteric Activation by AMP * ^♦

doi: 10.1074/jbc.M116.757062

Figure Lengend Snippet: Cys318 and Cys326 are critical for H2O2-dependent inhibition of bGP and may form an intramolecular disulfide bond in bGP. a, bGP sequence representation with relative positions of the cysteine residues. Four cysteine residues are specific from the brain isoform (C326*, C436*, C757*, and C808*). In addition, three cysteine residues have been described previously as reactive cysteine (colored in red) (22, 23, 26). Sequences of muscle (PYGM), liver (PYGL), and brain (PYGB) glycogen phosphorylase from rat, mouse, and human containing the three reactive cysteines have been aligned. b, ribbon representation of Cα trace of the dimer of bGP. The AMP-binding site is marked by the allosteric effector AMP (surface representation). The 12 cysteine residues are represented as a yellow stick. The distance separating the Cα of each pair of cysteine residues was measured (supplemental Table 1). Two pairs of cysteines were identified with Cα1-Cα2 <10 Å and are represented (right and left panels): Cys318–Cys326 and Cys373–Cys445. c, single mutation and double mutations of Cys318 and Cys326 were performed and tested for oxidation resistance as described above. Results are expressed as the percentage of the control. Data represent mean values of three independent experiments ± S.D., ***, p < 0.001 when compared with control; ###, p < 0.001 when two non-control groups are compared. d, HEK293T cells were transiently transfected with either WT bGP vector or C318S/C326S mutant bGP vector. Cell medium was gradually replaced by medium without FCS, and cells were then exposed to H2O2 (0, 250, and 500 μm) for 20 min at 37 °C under 5% CO2. Whole-cell extracts were then assayed for endogenous glycogen phosphorylase activity and Western blotting analysis. Activities are expressed as the percentage of the control. Data represent mean values of three independent experiments ± S.D., ***, p < 0.001, **, p < 0.01, when compared with control; ###, p < 0.001 when two non-control groups are compared. e, plot of the relative activity of bGP as a function of the ratio of reduced to oxidized glutathione. bGP activity was measured after incubation of the recombinant bGP with increasing ratios of [GSH]2/[GSSG] (0–20,000 mm) for 18 h at 4 °C. Activity was expressed as the percentage of the control (reduced protein). The solid line is the theoretical fit to Equation 4. The calculated E°′bGP was −267 mV. Data represent mean values of three independent experiments ± S.D.

Article Snippet: The oligonucleotides used for the site-directed mutagenesis were purchased from Eurofins. pCMV6 carrying bGP cDNA (pCMV6- PYGB ) was purchased from OriGene Technologies, Inc. Antibodies raised against brain glycogen phosphorylase were obtained from Santa Cruz Biotechnology (SC-81751).

Techniques: Inhibition, Sequencing, Binding Assay, Mutagenesis, Control, Transfection, Plasmid Preparation, Activity Assay, Western Blot, Incubation, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: An Isozyme-specific Redox Switch in Human Brain Glycogen Phosphorylase Modulates Its Allosteric Activation by AMP * ^♦

doi: 10.1074/jbc.M116.757062

Figure Lengend Snippet: H2O2 exposure and Cys318–Cys326 disulfide bond formation impacts the AMP-dependent activation of bGP. A, bGP was incubated with 250 μm H2O2 for 30 min at 37 °C. The oxidized enzyme was then incubated with AMP or phosphorylase kinase prior to activity measurement. Phos. Ser, phosphorylated serine. Results are expressed as the percentage of the control. Data represent mean values of three independent experiments ± S.D., ***, p < 0.001 when compared with control; ###, p < 0.001 when two non-control groups are compared. B, analysis of the binding of AMP to reduced and oxidized bGP was performed by incubating the treated or untreated enzyme with 5 μm fluorescent mant-AMP. To ascertain that the fluorescence was due to binding of mant-AMP to the enzyme, increasing concentrations of AMP were added. The addition of increasing concentrations of AMP resulted in a loss of fluorescence. A. U., arbitrary units.

Article Snippet: The oligonucleotides used for the site-directed mutagenesis were purchased from Eurofins. pCMV6 carrying bGP cDNA (pCMV6- PYGB ) was purchased from OriGene Technologies, Inc. Antibodies raised against brain glycogen phosphorylase were obtained from Santa Cruz Biotechnology (SC-81751).

Techniques: Activation Assay, Incubation, Activity Assay, Control, Binding Assay, Fluorescence