Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: XCL1 siRNA treatment abolished anti-apoptotic effect of human Wharton's jelly-derived human mesenchymal stem cell (WJ-MSC). (a) Before coculturing with serum-starved C2C12 cells, WJ-MSCs were separately pretreated with two siRNAs of human XCL1 containing different sequences for 24 hours. Each siRNA-treated WJ-MSC was cocultured with serum-deprived C2C12 cells for 12 hours. Human-recombinant XCL1 was added to cocultured C2C12 cells with siRNA-treated WJ-MSC to monitor gain of function of XCL1. C2C12 cell images were taken at each condition. (b) Cell lysates were analyzed by Western blot using anti-poly ADP-ribose polymerase (PARP) antibody. Cleaved PARP fragments were monitored by densitometric analysis (*P < 0.05, n = 3). (c) Secreted XCL1 concentration in each conditioned media was measured by XCL1 ELISA kit (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Derivative Assay, Recombinant, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Induction of human XCL1 reduces apoptosis of the skeletal muscles in the zebrafish myopathy model. Twenty hours post fertilization (hpf) zebrafish embryos (a–d) and 36 hpf embryos were observed (e–h). (a,e) The apoptotic cells of control and adssl1 morphants or human XCL1-induced adssl1 morphants were detected by TUNEL assay, and the whole-mounted embryos were observed by fluorescent microscopy. (b,f) The boxed region of the embryos was photographed using confocal microscopy. (c,g) The representative confocal images show that the number of dead cells in adssl1 morphants was more than in control morphants, and induction of human XCL1 inhibited the increase of cell death in adssl1 morphants. (d,h) The cell death in the selected area (within five somites in the trunk) was quantified and presented in the scattered chart. A, anterior; P, posterior. a,e, bar = 100 µm; b,c,f,g, bar = 50 µm. d,h, Student's t-test: *P < 0.05, **P < 0.005, ***P < 0.001.

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Muscles, Control, TUNEL Assay, Microscopy, Confocal Microscopy

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Identification of XCL1 as a paracrine factor of Wharton's jelly-derived human mesenchymal stem cell (WJ-MSC) during coculture. Secreted proteins in the media collected from the experiments were measured using the RayBio Biotin Label-based Human Antibody. (a,b) Increased fold of spot intensity of XCL1, IL-3 R, and smad5 in cocultured cells as compared to WJ-MSC alone. (c) The conditioned media was collected from C2C12, WJ-MSC alone, and cocultured cells. Concentration of secreted XCL1 in each conditioned media was measured by XCL1 ELISA kit (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Recombinant XCL1 protein inhibited serum starvation-induced apoptosis of C2C12 cells. C2C12 cell was serum-deprived during culture both in the absence or presence of recombinant XCL1 proteins (1, 10, and 20 ng/ml) for 12 hours. (a) Fluorescence-activated cell-sorting (FACS) analysis of cells stained with annexin V/7-AAD. (b) Percentage of apoptotic cells by FACS analysis (*P < 0.05, n = 3). (c) Harvested cells were analyzed by Western blot analysis with anti-poly ADP-ribose polymerase (PARP) antibody. (d) PARP cleaved bands were analyzed by densitometry (*P < 0.05, n = 3). (e) Serum-deprived C2C12 cells were treated with recombinant XCL1 (8 ng/ml) in a time-dependent manner. (f) PARP cleavage was monitored by Western blot analysis (*P < 0.05, n = 3). (g) Serum-deprived C2C12 cells were treated with either PAN caspase inhibitor (z-VAD-FMK) or XCL1 protein in a dose-dependent manner for 24 hours. (h) Apoptotic cells were analyzed by Western blot with anti-PARP antibody. (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Recombinant, Fluorescence, FACS, Staining, Western Blot

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Absence of anti-apoptotic effect of XCL1 in other types of cells. The HT22, mouse hippocampal neuron cells were treated with amyloid-β42 (1 μmol/l for 48 hours) or MG132 (5 μmol/l for 24 hours) in the absence or presence of XCL1. (a) Fluorescence-activated cell-sorting (FACS) analysis of cells stained with annexin V/7-AAD. (b) Poly ADP-ribose polymerase (PARP)-cleaved bands were analyzed by densitometry. S16 rat Schwann cells were treated with thapsigargin (0.1 μmol/l for 12 hours) in the absence or presence of XCL1. The thapsigargin (0.1 μmol/l for 12 hours) treated S16 cells were analyzed by (c) FACS analysis and (d) Western blot analysis with anti-PARP antibody.

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Fluorescence, FACS, Staining, Western Blot

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: XCL1 treatment inhibited lovastatin-induced apoptosis of C2C12 cells and reduction of myotube formation. (a) C2C12 cells were treated with lovastatin (0.1, 0.5, 1, and 2 μmol/l) in a dose-dependent manner in the absence or presence of XCL1 and protein lysates were analyzed by Western blot analysis using anti-poly ADP-ribose polymerase (PARP) antibody. (b) Cleaved PARP fragments were monitored by densitometric analysis (*P < 0.05, n = 3). (c) The 7-day differentiated myotubes from C2C12 cells were treated with lovastatin (1, 2.5, 5, and 10 μmol/l) in the absence or presence of XCL1. Each arrow indicates defects in the myotubes. (d,e) Protein extracts of myotubes were analyzed by Western blot analysis using anti-myosin heavy chain (MHC) antibody. Bands of MHC were monitored by densitometric analysis (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Western Blot

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: The rescue of defective skeletal muscles by human XCL1 in the zebrafish model. (a) The boxed region indicates the observed area to determine the effect of human XCL1 on skeletal muscles in zebrafish at 36 hpf. Myosepta and muscle fibers were stained with anti-Laminin and myosin heavy chain (MHC) antibodies, respectively. (b) The whole-mounted embryos after double staining with anti-Laminin and MHC antibodies were observed by confocal microscopy. The adssl1 morphants (phosphate-buffered saline injected embryos) showed disrupted muscle fibers and myosepta, whereas the morphants injected with human XCL1 showed recovery of muscle defects. (c) The zebrafish embryos demonstrating skeletal muscle phenotype were counted and quantified data were presented graphically. The data indicates that human XCL1 restores muscle abnormality of adssl1 morphants in a dose-dependent manner. A, anterior; P, posterior. Scale bar = 50 µm.

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Muscles, Staining, Double Staining, Confocal Microscopy, Saline, Injection

Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: Primers used in the real-time PCR

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: Examination of tissue and cell expression of IL-32 by quantitative real-time PCR. (a) Tissue expression of IL-32. WBC, white blood cells. (b) Human peripheral blood mononuclear cells (PBMCs) expressed IL-32. PBMCs were cultured with or without concanavalin A. PBMCs were also stimulated by immobilized anti-human CD3 and anti-human CD28 antibodies. Cont, control. (c) IL-32 expression of monocytes and B cells after the depletion of CD3 + cells. (d) Peripheral CD4 + T cells were cultured with the indicated inflammatory cytokines for 24 hours. (e) Human monocyte-derived dendritic cells (MoDCs) were cultured with lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNFα) for 24 hours to induce maturation. The data are representative of at least three independent studies.

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Control, Derivative Assay

Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: IL-32 was abundantly expressed in the synovial tissues of rheumatoid arthritis patients. (a) In situ hybridization of the synovial tissues from rheumatoid arthritis (RA) patients. IL-32β was expressed in the synovial-infiltrated lymphocytes of RA patients. HE stain, hematoxylin and eosin stain. We examined the tissue samples from four RA patients, and show representative examples. (b) IL-32 expression of the synovial fibroblasts derived from four RA patients in response to human tumor necrosis factor alpha (hTNFα).

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: In Situ Hybridization, H&E Stain, Expressing, Derivative Assay

Journal: Arthritis Research & Therapy

Article Title: Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

doi: 10.1186/ar2074

Figure Lengend Snippet: Inflammatory cytokines were induced by human IL-32β in mice. (a) Raw 267.4 was cultured with the supernatant of human IL-32β (h IL-32β) or mock-transfected mammalian cells (293T) for 24 hours. Left, relative expression of mouse tumor necrosis factor alpha (mTNFα), compared with β-actin; right, secreted TNFα protein level measured by ELISA. (b) We generated hIL-32β overexpressed mice by transplantation of hIL-32β-transduced bone marrow cells. The expression of green fluorescent protein, was analyzed by flow cytometry 6–9 weeks after transplantation. (c) Expression of mTNFα, mIL-1β and mIL-6 in the cultured splenocytes of the control group (white bars; n = 3), or bone-marrow chimeric mice of the mock group (BM-Mock mice) (gray bars; n = 4), or hIL-32β (BM-hIL-32) (black bars; n = 4) with or without 1 μg/ml lipopolysaccharide (LPS). Concentrations of indicated cytokines of the cultured supernatants are shown in the right-hand figures. (d) Serum concentration of mTNFα determined in control mice ( n = 4), in BM-Mock mice ( n = 8), and in BM-hIL-32 mice ( n = 8). (e) Expression of mTNFα in splenic F4/80 + CD11c - macrophages of BM-Mock mice (gray bars; n = 4) and in BM-hIL-32 mice (black bars; n = 4). (f) Expression of mTNFα, mIL-1β, and mIL-6 in LPS-stimulated splenic F4/80 + CD11c - macrophages and CD11c + , CD3 - , and CD19 - dendritic cells in BM-Mock mice (gray bars; n = 4), and in BM-hIL-32 mice (black bars; n = 4). Data are representative of at least three independent studies. * P < 0.05, ** P < 0.01, BM-hIL-32 mice versus BM-Mock mice or control mice.

Article Snippet: Recombinant human TNFα, IL-12, IL-18, IL-23, granulocyte-macrophage colony-stimulating factor, and IL-4 were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Generated, Transplantation Assay, Flow Cytometry, Control, Concentration Assay

Journal: Nature Communications

Article Title: Pancreatic tumor eradication via selective Pin1 inhibition in cancer-associated fibroblasts and T lymphocytes engagement

doi: 10.1038/s41467-022-31928-7

Figure Lengend Snippet: a Establishment and validation of CAFs via high expressions of both FAP-α and α-SMA. b Cellular uptake of Cy5-labeled formulations on Pan02 cells, NIH-3T3 cells, and CAFs, measured by flow cytometry ( n = 3 biologically independent experiments; error bars, mean ± SD). c Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, imaged by fluorescent microscopy. Scale bars are 20 μm. Three independent repetitions got similar results. d Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, quantified by flow cytometry ( n = 6 biologically independent samples; error bars, mean ± SD). Cytotoxicity on Pan02 cells ( e ) and CAFs ( f ) ( n = 3 biologically independent experiments; error bars, mean ± SD). g Western blotting detection of Pin1 and relative proteins from Pan02 cells after treating with PBS, AG17724 (0.5 μM), or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. h Quantitative analysis of the western blotting results from Pan02 cells (Three independent repetitions got similar results.). i Western blotting detection of Pin1 and relative proteins from CAFs treated with PBS, AG17724, or antiCAFs-DMS after treating with PBS, AG17724 (0.5 μM) or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. j Quantitative analysis of the western blotting results from CAFs (Three independent repetitions got similar results.). One-way analysis of variance followed by Turkey posttests. Source data are provided as a Source Data file.

Article Snippet: Anti-FAP-α antibody (MAB9727-100, 0.25 μg/mL as working concentration.) and anti-phospho-CDC2/CDK1 (Y15) antibody (AF888-SP, 0.2 μg/mL as working concentration.) were purchased from R&D SYSTEMS.

Techniques: Biomarker Discovery, Labeling, Flow Cytometry, Microscopy, Western Blot

Journal: Nutrition & Metabolism

Article Title: Zinc, vitamin A, and vitamin C status are associated with leptin concentrations and obesity in Mexican women: results from a cross-sectional study

doi: 10.1186/1743-7075-9-59

Figure Lengend Snippet: Age, anthropometry, body composition, and biochemical variables of the women that participated in the study 1

Article Snippet: Plasma leptin concentration was quantified by a commercial kit using ELISA (Human Leptin Elisa Kit, Linco Research, St Charles, MO) using a microplate photometer (Multiskan Ascent, Thermo Electron Corporation, Vaanta, Finland).

Techniques: Fat

Journal: Nutrition & Metabolism

Article Title: Zinc, vitamin A, and vitamin C status are associated with leptin concentrations and obesity in Mexican women: results from a cross-sectional study

doi: 10.1186/1743-7075-9-59

Figure Lengend Snippet: Association of zinc and vitamins C, A and E concentrations with leptin concentration, body fat and anthropometry of the women that participated in the study 1

Article Snippet: Plasma leptin concentration was quantified by a commercial kit using ELISA (Human Leptin Elisa Kit, Linco Research, St Charles, MO) using a microplate photometer (Multiskan Ascent, Thermo Electron Corporation, Vaanta, Finland).

Techniques: Concentration Assay, Fat

Journal: Nutrition & Metabolism

Article Title: Zinc, vitamin A, and vitamin C status are associated with leptin concentrations and obesity in Mexican women: results from a cross-sectional study

doi: 10.1186/1743-7075-9-59

Figure Lengend Snippet: Association of leptin concentration with vitamins A, C, E and zinc in the women that participated in the related to Body Mass Index (BMI), body fat and waist circumference terciles 1

Article Snippet: Plasma leptin concentration was quantified by a commercial kit using ELISA (Human Leptin Elisa Kit, Linco Research, St Charles, MO) using a microplate photometer (Multiskan Ascent, Thermo Electron Corporation, Vaanta, Finland).

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

doi: 10.3390/ijms22031105

Figure Lengend Snippet: ( A ) Western Blot analysis of proteins present (Hsp90, CD63 and TSG101) or absent (calnexin) in exosomes and abundant in bovine milk (β-casein). Protein evaluation in: bovine skimmed milk (SM); exosomes obtained from SM by one (1U) or two (2U) ultracentrifugation steps; and the cellular fraction (CF). Equal amount of protein was loaded. Elution protein profile (F.1, F.8 to F.40) of bovine exosomes isolated by 1U followed by SEC (1U + SEC) ( B ) or 2U + SEC ( C ). Protein concentration (mg/mL) was estimated by the BCA assay. WB of SEC elution fractions from exosomes isolated by 1U + SEC ( D ) or 2U + SEC ( E ). Evaluation of Hsp90, CD63, TSG101, Calnexin and β-casein levels in each fraction (F.1, F.8 to F.40). Mimic hsa-miRNA-148a-3p elution profile (relative expression) of exosomes isolated from skimmed milk by U + T + SEC ( F ) or U + T + U + SEC ( G ). Mw: Molecular weight marker (Bio-Rad).

Article Snippet: Membranes were then incubated with appropriate primary antibodies: anti-Hsp90 (610418, BD, Madrid, Spain), anti-CD63 (bs-1523R, BIOSS, Woburn, MA, USA), anti-TSG101 (A303-506A, Bethyl, Montgomery, TX, USA), anti-calnexin (A303-694A, Bethyl) or anti-β-casein (ab112595, abcam, Cambridge, UK) at 4 °C (overnight).

Techniques: Western Blot, Isolation, Protein Concentration, BIA-KA, Expressing, Molecular Weight, Marker

Journal: Gels

Article Title: Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

doi: 10.3390/gels9030243

Figure Lengend Snippet: The production yield, activity, and purification of recombinant Cripto produced in the 3D microcarriers as compared to the 2D method. ( A ) SDS-PAGE analysis of the purification steps of recombinant Cripto: lane M is the protein molecular weight marker; lane 1 is the protein solution from ultrafiltration; lane 2 is the flowthrough after the first passage through the His-tag affinity Ni-NTA resin; lane 3 is the flowthrough after the second passage through the same resin; lane 4 is the first wash step; lane 5 is the second wash step; lane 6 is the first elution from the His-tag affinity Ni-NTA resin; and lane 7 is the second elution from the same resin. The band at approximately 27 kDa is the Cripto protein (indicated by the red arrow). ( B ) Quantitative amounts of Cripto protein produced in the 3D batch (Cripto (3D) ) with HEK293 cells encapsulated in PF microcarriers and incubated in bioreactors after three rounds of harvesting are compared to the maximum amount of Cripto produced in the 2D batch (Cripto (2D) ) with HEK293 cells adherent to cell culture plates and cultured to their density threshold limits. An initial cell seeding of 3.2 × 10 6 cells was used for both techniques. ( C ) The biological activity of recombinant Cripto was compared for Cripto produced in PF microcarriers versus the 2D method by measuring binding affinity to the AlK4 receptor. Four independent experiments were carried out for the 3D system and three independent experiments were performed for the 2D cultivation method. Results are shown as mean ± S.D. *** indicates p < 0.001.

Article Snippet: Serial dilutions of the concentrated protein were put into 96-well plates coated with either mouse Cripto antibody (for total concentration) or recombinant mouse activin receptor IB/Fc (for active protein concentration) (R&D systems AF1538 and 1477-AR, respectively).

Techniques: Activity Assay, Purification, Recombinant, Produced, SDS Page, Molecular Weight, Marker, Incubation, Cell Culture, Binding Assay

Journal: Gels

Article Title: Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

doi: 10.3390/gels9030243

Figure Lengend Snippet: The effect of Cripto produced in 3D microcarriers on C2C12 cell proliferation detected by the BrdU incorporation assay. ( A ) C2C12 myoblast proliferation was evaluated by the BrdU incorporation assay after being cultured for 48 h in serum-free medium containing Cripto produced in 3D microcarriers (Cripto (3D) ) or commercially available Cripto (Cripto (R&D) ). Also evaluated were a bFGF medium positive control and a serum-free medium negative control. ( B ) The recombinant Cripto induces myoblast proliferation in a dose-dependent pattern; increasing concentrations of Cripto (3D) and Cripto (R&D) were added to C2C12 cells, and proliferation was quantified by the BrdU incorporation assay. ( C ) Cell proliferation was further evaluated by counting the total number of live cells. The proliferative effect of Cripto (3D) was compared with that of commercial Cripto (R&D) . The data are presented as mean ± S.D. from at least three independent experiments. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: Serial dilutions of the concentrated protein were put into 96-well plates coated with either mouse Cripto antibody (for total concentration) or recombinant mouse activin receptor IB/Fc (for active protein concentration) (R&D systems AF1538 and 1477-AR, respectively).

Techniques: Produced, BrdU Incorporation Assay, Cell Culture, Positive Control, Negative Control, Recombinant

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Bacterial Strains and Plasmid Constructs used in this study.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Plasmid Preparation, Construct, Control, Knock-In, Over Expression, Recombinant

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: B. miyamotoi encodes two orthologous genes to B. burgdorferi BBK32. (A) BBK32 orthologs are found in relapsing fever (RF)-associated and B. miyamotoi spirochetes and are denoted FbpA, FbpB, and FbpC . B. miyamotoi FR64b FbpA and FbpB are underlined. (B) An alignment of B. miyamotoi strain FR64b FbpA and FbpB to B. burgdorferi strain B31 BBK32 shows differences of the amino acid sequences within the fibronectin binding (green box) and complement inhibitory domains (blue box). The gelatin-binding domain (GBD) of BBK32 is denoted. The key residues R248 and K327 of BBK32 involved in complement C1r binding are indicated by a red box. * conserved, : strongly similar, . weakly similar.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Assessing the interaction of human C1r with B. miyamotoi FbpA and FbpB. SPR was used to assess protein-protein interactions between the C-terminal regions of each Fbp protein and activated C1r-CCP2-SP. Immobilized (A) FbpA-C, (B) FbpB-C, and (C) FbpA-C-R264A-K343A (referred to as FbpA DA-C throughout) were subjected to an injection series of serially diluted C1r-CCP2-SP (0.78 - 100 nM). A representative sensorgram from the three replicates for each Fbp is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D values were determined using kinetic fits and are shown as the mean +/- standard deviation of three replicates in Table 4 . A K D value for C1r-CCP2-SP interactions with FbpA DA-C was not determined (N.D.). (D, E) Competition experiments were carried out using 25 nM C1r-CCP2-SP alone or mixed with 25 nM soluble Fbp proteins injected over FbpA-C (D) or FbpB-C (E) . (F) Comparisons of statistical significance were performed for data shown in panels (D, E) using a one-way ANOVA followed by a multiple comparison Tukey test (* = p < 0.05).

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Protein-Protein interactions, Injection, Standard Deviation, Comparison

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: SPR and complement assay results.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Complement Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: FbpA-C and FbpB-C interact differentially with zymogen and activated forms of human C1r. (A) Single cycle SPR was used to determine binding affinities of C1r zymogen or active C1r ranging from 0.16 - 100 nM injected over immobilized Fbps. A representative sensorgram from a three-injection series for each Fbp-C and C1r active state is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D ’s were determined using kinetic fits and are shown as the average and standard deviation of three replicates in Table 4 . (B) Far Western overlays using B. burgdorferi B314 lysates probed with zymogen or enzymatic forms of purified full-length C1r, followed by an antibody to C1r. (C) An ELISA-type binding assay was used to determine the ability of FbpA-C, FbpA DA-C and FbpB-C to interact with C1r in human serum. FbpA-C (black), FbpB-C (blue), and FbpA DA-C (red) were immobilized on an ELISA plate then incubated with a two-fold dilution series of normal human serum ranging from 0.0012-10%. Corresponding EC 50 values for FbpA-C, FbpB-C, and FbpA DA-C are reported in Table 4 . (D) Fbp-mediated inhibition of purified active C1r-CCP2-SP enzymatic activity was assessed by incubating 15 nM C1r-CCP2-SP with a 1 μM of FbpA-C (white), FbpB-C (striped), and FbpA DA-C (grey). C1r-CCP2-SP enzymatic activity was determined by incubation with a C1r substrate (Z-Gly-Arg-sBzl) that, once cleaved, reacts with DTNB resulting in a colorimetric change. No statistical difference was found between FbpA-C and FbpB-C inhibitory activity, whereas FbpA DA-C exhibited significant loss in inhibitory activity. Statistical analysis was performed using a one-way ANOVA followed by a multiple comparison Tukey test (*=p<0.05).

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Binding Assay, Injection, Standard Deviation, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Inhibition, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Determining the ability of surface-expressed B. miyamotoi FbpA and FbpB to protect a serum-sensitive strain of B. burgdorferi . (A) Western blots of lysates from B. miyamotoi strain FR64b and B burgdorferi B314 isolates expressing fbpA , fbpA-DA , fbpB , and bbk32 were probed with antibodies to FbpA (anti-FbpA), FbpB (anti-FbpB), BBK32 (anti-BBK32) and FlaB (anti-FlaB), the latter as a loading control and a control for a subsurface target. Vector refers to the plasmid-only backbone sample (B314/pBBE22 luc ). Samples from each population were subjected to proteinase K accessibility treatments to determine the surface expression of the proteins. Due to its periplasmic location, the flagellar protein, FlaB, is unaffected by proteinase K in intact cells and depicts structural integrity in the treated B. miyamotoi and B314 derivatives listed. (B) B. miyamotoi Fbp proteins were tested for their ability to confer resistance to normal human serum (NHS) sensitive B. burgdorferi strain B314 relative to vector-only and BBK32-expressing controls (negative and positive controls, respectively). Asterisks depict a significant increase in survival relative to FbpA DA and the vector control (*=p<0.0001). All strains exposed to heat inactivated NHS (hiNHS), rendering complement proteins inactive, were largely unaffected. (C) Rescue assays were used to determine if soluble exogenous recombinant proteins could promote survival of serum-sensitive vector-containing B. burgdorferi B314. Increasing five-fold concentrations of BBK32-C, FbpA-C, FbpA DA-C, and FbpB-C were added in serum sensitivity assays. The middle samples (i.e., 240 nM) have added recombinant protein that is roughly equivalent to the concentration of C1r in the assay conditions employed. Cells were assessed via darkfield microscopy. Included as controls are a vector-only strain of B314 with no exogenous protein addition, as well as a BBK32-producing B314 isolate (right side). Asterisks depict a significant increase in survival relative to the vector control (*=p<0.015). Significance for serum sensitivity and rescue assays was determined using two-way ANOVA with a Šidák correction for multiple comparisons.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Recombinant, Concentration Assay, Microscopy