Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Adult ovine chondrocytes in expansion culture adopt progenitor cell properties that are favorable for cartilage tissue engineering

doi: 10.1002/jor.24671

Figure Lengend Snippet: Cumulative population doubling of ovine chondrocytes expanded in DMEM or αMEM basal medium. Expansion media were supplemented with 10% FBS and 2 ng/mL FGF-2. Experiments were conducted using chondrocytes from three animals (N = 3). DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FGF-2, fibroblast growth factor 2

Article Snippet: 21 Chondrogenic medium consisted of high glucose DMEM (Thermo Fisher Scientific plus 10mM HEPES, 0.5% antibiotics/antimycotics solution, 2% FBS, 1% ITS+Premix (BD Biosciences, Bedford, MA), 37.5 μg/mL ascorbate-2-phosphate (Wako Chemicals, Richmond, VA), 100 nM dexamethasone (Dex) (Sigma-Aldrich, Saint Louis, MO) and 5 ng/mL recombinant human transforming growth factor beta 1 (TGFβ) (Peprotech).

Techniques: Modification

Journal: Diabetologia

Article Title: Exercise as a non-pharmacological intervention to protect pancreatic beta cells in individuals with type 1 and type 2 diabetes

doi: 10.1007/s00125-022-05837-9

Figure Lengend Snippet: Recombinant clusterin protects human beta cells against thapsigargin-induced apoptosis. EndoC-βH1 cells were pretreated for 24 h with 0 (white bars), 1 (grey bars) or 100 ng/ml (purple bars) recombinant clusterin. Medium was then changed, and cells were exposed or not to clusterin and thapsigargin (THAP, 1 μmol/l). Apoptosis ( a ) and DP5 expression ( b ) were analysed after 48 h. Gene expression was corrected for the geometric mean of the reference genes ACTB and VAPA and normalised to the condition THAP without clusterin set as 1. Results are mean ± SEM of six independent experiments. ** p <0.01 and *** p <0.001 compared with no clusterin/non-treated (NT); †† p <0.01 and ††† p <0.001 compared with no clusterin/THAP (ANOVA)

Article Snippet: EndoC-βH1 cells were pre-cultured for 24 h in medium supplemented with 2% FBS plus recombinant clusterin (no. 2937-HS-050, R&D Systems, Minneapolis, MN, USA) at concentrations of 1 and 100 ng/ml.

Techniques: Recombinant, Expressing, Gene Expression

Journal: The Journal of Experimental Medicine

Article Title: APRIL, a New Ligand of the Tumor Necrosis Factor Family, Stimulates Tumor Cell Growth

doi:

Figure Lengend Snippet: ( A ) Predicted amino acid sequence of human APRIL. The predicted transmembrane region ( TM , boxed ), the potential N-linked glycosylation site (*), and the NH 2 terminus of the recombinant sAPRIL are indicated. ( B ) Comparison of the extracellular protein sequence of APRIL and some members of the TNF ligand family. Identical and homologous residues are represented in black and shaded boxes, respectively. TNFa , TNF-α; LTa , LTα.

Article Snippet: The proliferation of cells was determined by incubating 5 × 10 4 cells/well in 100 μl medium with the indicated concentrations of recombinant sAPRIL, sTWEAK, and sFasL using the Celltiter 96 AQ proliferation assay ( Promega Corp. , Madison, WI) after 24 h, following the manufacturer's instructions.

Techniques: Sequencing, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: APRIL, a New Ligand of the Tumor Necrosis Factor Family, Stimulates Tumor Cell Growth

doi:

Figure Lengend Snippet: APRIL stimulates cell growth. ( A ) Dose-dependent stimulation of proliferation of Jurkat cells (human leukemic T cells), determined 24 h after the addition of sAPRIL. Controls include cells treated with FasL, TWEAK, and no ligand ( Control ). Left , Number of viable cells; middle , [ 3 H]thymidine incorporation; right , kinetic analysis of the effect of APRIL on Jurkat cells. The concentrations of ligands are indicated. ( B ) Effect of APRIL on the proliferation rate of Raji (human Burkitt lymphoma), A20 (mouse B lymphoma), BJAB (human B lymphoma), COS (SV40-transformed monkey kidney cells), MCF-7 (human breast adenocarcinoma), HeLa (human embryonic lung) cell, and ME260 (human melanoma). ( C ) Influence of immunodepletion of FLAG-tagged APRIL on tumor cell growth. The proliferative effect of FLAG-tagged APRIL is neutralized by Sepharose-bound anti-FLAG antibodies, but not by anti-myc antibodies. ( D ) Influence of FCS concentration on APRIL-induced proliferation of Jurkat cells. Data are the means ± SEM of triplicate determinations.

Article Snippet: The proliferation of cells was determined by incubating 5 × 10 4 cells/well in 100 μl medium with the indicated concentrations of recombinant sAPRIL, sTWEAK, and sFasL using the Celltiter 96 AQ proliferation assay ( Promega Corp. , Madison, WI) after 24 h, following the manufacturer's instructions.

Techniques: Transformation Assay, Concentration Assay

Journal: Journal of Virology

Article Title: Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5? Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome ^▿

doi: 10.1128/JVI.00339-11

Figure Lengend Snippet: Identification of PCBP2 binding to HCV 5′UTR and 3′UTR by streptavidin-biotin RNA-protein binding assay. (A) Huh7 cytoplasmic extract was incubated with various biotin-labeled RNA and selected with streptavidin beads. Proteins binding to the RNA were blotted with mouse anti-PCBP2 antibody. (B) PCBP2 bound to the fragments containing nt 1 to 157 of the HCV 5′UTR, SL1, and the 157-nt fragment deleting SL2, but it did not bind to SL2 or the 157-nt fragment deleting SL1. (C) PCBP2 also bound to the 3′ end of HCV RNA, but it did so much more weakly than to the 5′ end.

Article Snippet: The 32 P-labeled RNA probe was incubated with bovine serum albumin (BSA) or recombinant PCBP2 (Abnova) in the presence of 10 μg of tRNA.

Techniques: Binding Assay, Protein Binding, Incubation, Labeling

Journal: Journal of Virology

Article Title: Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5? Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome ^▿

doi: 10.1128/JVI.00339-11

Figure Lengend Snippet: Effects of PCBP2 knockdown in HCV replicon cells and HCV-infected cells. (A) The HCV replicon Rep1.1 cells were infected with lentiviruses containing four different shPCBP2 clones. After puromycin selection for 3 days, cell lysates were used for immunoblotting with different antibodies to detect the various proteins. PC (positive control) stands for the replicon cell lysates without the lentivirus infection. (B) The HCV replicon cells were infected with shPCBP2 clones A and B and unrelated shLacZ and then incubated in the presence of puromycin for different days. The RNAs harvested at different time points were quantified by quantitative reverse transcription-PCR (qRT-PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. (C) Huh7.5 cells were infected with lentiviruses containing shPCBP2 (clones A and B) or shLacZ. After puromycin selection for 2 days, the cells were infected with JC1 virus. At 3 days after JC1 virus infection, intracellular proteins and RNAs were analyzed by immunoblotting (C) and qRT-PCR (D), respectively.

Article Snippet: The 32 P-labeled RNA probe was incubated with bovine serum albumin (BSA) or recombinant PCBP2 (Abnova) in the presence of 10 μg of tRNA.

Techniques: Knockdown, Infection, Clone Assay, Selection, Western Blot, Positive Control, Incubation, Reverse Transcription, Quantitative RT-PCR, Control, Virus

Journal: Journal of Virology

Article Title: Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5? Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome ^▿

doi: 10.1128/JVI.00339-11

Figure Lengend Snippet: (A) Membrane flotation analysis of the location of PCBP2. Cell lysates from Huh7 and replicon cells left untreated or treated with Triton X-100 were separated by sucrose gradient sedimentation as described in Materials and Methods. Each fraction was blotted with various antibodies. Fractions 2 to 4 represent the membrane fraction. Fractions 5 to 9 represent the cytosolic fraction. (B) The immunofluorescence of PCBP2 in the naïve Huh7 cells and replicon cells. (C) The nuclear and cytoplasmic extracts of Huh7 and replicon cells were separated and blotted with PCBP2 antibody. (D) Immunoprecipitation assay of the HCV viral proteins interacting with PCBP2. Plasmids expressing NS3/4A (pCI-NS3/4A-HA), NS4B (pUI-NS4B-HA), NS5A (pUI-NS5A-HA), NS5B (pCAG2-NS5B-HA), and EYFP (pCAG-EYFP-HA), all carrying an HA tag, were transfected into 293T cells individually. The cell lysates were immunoprecipitated with rabbit anti-PCBP2 antibody, and each NS protein was detected with mouse anti-HA antibody by immunoblotting.

Article Snippet: The 32 P-labeled RNA probe was incubated with bovine serum albumin (BSA) or recombinant PCBP2 (Abnova) in the presence of 10 μg of tRNA.

Techniques: Membrane, Sedimentation, Immunofluorescence, Immunoprecipitation, Expressing, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5? Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome ^▿

doi: 10.1128/JVI.00339-11

Figure Lengend Snippet: Antibody-mediated blocking of PCBP2 repressed HCV replication in in vitro replication assay. HCV replicon cell lysates were incubated with increasing amounts of anti-calnexin, anti-VAP-33, or anti-PCPB2 antibodies to block the indicated proteins. The samples then were incubated with [α-32P]UTP in a cell-free RNA-dependent RNA polymerase assay. The HCV RNA product was detected by autoradiography after separation by formaldehyde agarose gel electrophoresis. In vitro-transcribed replicon RNA served as a marker to indicate the size of HCV replicon RNA.

Article Snippet: The 32 P-labeled RNA probe was incubated with bovine serum albumin (BSA) or recombinant PCBP2 (Abnova) in the presence of 10 μg of tRNA.

Techniques: Blocking Assay, In Vitro, Incubation, Autoradiography, Agarose Gel Electrophoresis, Marker

Journal: Journal of Virology

Article Title: Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5? Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome ^▿

doi: 10.1128/JVI.00339-11

Figure Lengend Snippet: PCBP2 is involved in HCV IRES-mediated translation. (A) The structure of the bicistronic plasmid psiCHECK containing renilla and firefly luciferase genes mediated by cap or IRES-dependent translation, respectively. (B) Huh7 cells were infected with shLacZ or shPCBP2, and 3 days later they were transfected with psiCHECK plasmid. Cell lysates were collected at 20 h after transfection, and a luciferase assay was performed. HCV IRES activity relative to the cap-dependent translation was calculated based on the two luciferase activities.

Article Snippet: The 32 P-labeled RNA probe was incubated with bovine serum albumin (BSA) or recombinant PCBP2 (Abnova) in the presence of 10 μg of tRNA.

Techniques: Plasmid Preparation, Luciferase, Infection, Transfection, Activity Assay

Journal: Journal of Virology

Article Title: Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5? Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome ^▿

doi: 10.1128/JVI.00339-11

Figure Lengend Snippet: 5′-3′UTR coprecipitation assay. (A) Schematic sketch of the experimental protocol (see Materials and Methods for details). (B) Autoradiographs of the RNAs bound to the streptavidin beads using different combinations of proteins and RNA fragments as indicated. BSA was used as an unrelated protein. Increasing amounts of unlabeled 5′UTR and 3′UTR, but not the unrelated RNA, competed away the binding of 32P-3′UTR to bio-5′UTR. (C) SL1, but not SL2, of the HCV 5′UTR binds to the 3′UTR in the presence of PCBP2.

Article Snippet: The 32 P-labeled RNA probe was incubated with bovine serum albumin (BSA) or recombinant PCBP2 (Abnova) in the presence of 10 μg of tRNA.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5? Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome ^▿

doi: 10.1128/JVI.00339-11

Figure Lengend Snippet: HCV RNA circularizes in the presence of PCBP2. (A) Scheme of the structure of RNA molecules used (see Materials and Methods). (B) RNA electron micrographs. RNA molecules with 5′- and/or 3′UTRs were incubated with PCBP2 or BSA and observed under EM. One hundred molecules were counted for each set, and the ratio of circular to linear RNA molecules is indicated. Arrows indicate protein molecules binding at the end of RNA or on the circularized RNA. Bar, 200 nm.

Article Snippet: The 32 P-labeled RNA probe was incubated with bovine serum albumin (BSA) or recombinant PCBP2 (Abnova) in the presence of 10 μg of tRNA.

Techniques: Incubation, Binding Assay