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 oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 1 | CRIPTO (CR-1) is dynamically expressed in stem cell-enriched cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC). (A) Hematoxylin/Eosin staining of paraffin-embedded xenograft sections obtained from two SCC and two AC spheroid cultures (upper panels) and of the parental patient tumor (lower panels), 20X magnification, scale bar 50 mm. Pictures in the upper panels are representative of xenograft sections derived from three different mice. (B) Flow cytometry analysis of surface CRIPTO expression on two SCC and two AC spheroid cultures derived from patients represented in (A) and in Table S1. (C) Immunofluorescence staining for CRIPTO in combination with E-Cadherin or Prolyl 4-Hydroxylase subunit beta (P4HB) to show respectively plasmamembrane and endoplasmic reticulum localization on AC1 cells, 60X magnification, 1.8X zoom, scale bar 10 mm. (D) Values obtained from flow cytometry analysis of surface CRIPTO (indicated as percentage of positive cells) in AC1 (light purple rhombus) and SCC1 (light blue square) at the indicated days. The histogram represents the percentage of FACS-positive cells for 7-amino actinomycin D (7-AAD). FACS plots are shown in Supplemental Figure 1.

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Staining, Derivative Assay, Flow Cytometry, Expressing, Cytometry

Journal: Frontiers in oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 2 | CRIPTO (CR-1) expression regulates cells proliferation and stem cell gene expression in NSCLC spheroids. (A) Immunoblot analysis of CRIPTO and bActin in SCC1 spheroids transduced with the empty vector (Vector), with CRIPTO shRNA vector (CRIPTO KO) or with exogenous CRIPTO (CRIPTO-over). (B) CRIPTO and stem cell genes (OCT3/4, SOX2, NANOG) mRNA expression in SCC1 transduced with vector, CRIPTO KO or CRIPTO-over sequences. (C) ATP assay performed on SCC1 transduced with the vector, CRIPTO KO or CRIPTO-over sequences, performed 4 days after transduction (D). Cell cycle analysis of SCC1 transduced as above, performed 3 days after transduction. (E) Soft agar pictures (left; two technical replicates for each sample) and graph (right) of colony forming assay performed on control (Vector), CRIPTO KO and CRIPTO overexpressing SCC1 spheroids. The results are evaluated as colony formation in semisolid culture and expressed as normalized colony size/percentage over plated cells. (F) ELISA assay performed on SCC1 cells transduced with empty vector, CRIPTO KO sequences and CRIPTO overexpression vector. (G) Invasion/migration assay performed on vector-transduced, CRIPTO KO and CRIPTO overexpressing SCC1 cells. *P < 0.05; **P < 0.01, ***P < 0.001 by unpaired student’s t test (transduced vs vector).

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, Gene Expression, Western Blot, Transduction, Plasmid Preparation, shRNA, ATP Assay, Cell Cycle Assay, Control, Enzyme-linked Immunosorbent Assay, Over Expression, Migration

Journal: Frontiers in oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 3 | NSCLC subpopulations expressing high or low CRIPTO (CR-1) levels are interconvertible in vitro and in vivo. (A) FACS-based separation of CRIPTOhigh

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, In Vitro, In Vivo

Journal: Frontiers in oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 4 | Chemotherapy treatment increases CRIPTO (CR-1) expression and tumor progression in NSCLC xenografts. (A) Flow cytometry analysis of CRIPTO on SCC1 cells treated with vehicle only (Vehicle) or with chemotherapeutic agents (Cis+Gem early and Cis+Gem late). Cells in the Cis+Gem samples were treated with Cisplatin 5 mM plus Gemcitabine 25 mM for 4 days then washed, replated and analyzed after 3 additional days (Cis+Gem early) or 7 additional days (Cis+Gem late). The graph shows the mean ± SD of two independent experiments. Ns, non-significant, ***P < 0.001. (B) Left: Xenograft volume of SCC1 spheroids cells treated with Vehicle (Vehicle, light blue square) or with Cisplatin plus Gemcitabine (Cis+Gem, Dark pink dots). Mean ± SEM, 6 mice/group. **P < 0.01 (two-tailed t test). Middle: representative confocal images of CRIPTO (red) and TUNEL (green) staining of Vehicle and Cis+Gem treated tumors. 40X magnification, scale bar 50 mm. Right: quantification of CRIPTO and TUNEL performed on 3 sets composed of 5 fields/group. **P < 0.01. AU, arbitrary units. (C) Tumor variation after treatment withdrawal of SCC1 spheroids treated with vehicle (Vehicle, light blue square), and Cis plus Gemcitabine (Cis+Gem, dark pink dots). Mean ± SEM, 4 mice/group. (D) qRT-PCR analysis of CRIPTO and Cyclin E mRNA expression of SCC1-derived xenografts, monitored during treatment and after treatment withdrawal at the indicated times. *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, Flow Cytometry, Two Tailed Test, TUNEL Assay, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway.

doi: 10.4049/jimmunol.1202451

Figure Lengend Snippet: FIGURE 1. Characterization of the recom- binant CR1 CCP 22–30_His fragment. (A) SDS-PAGE analysis of 4 mg CR1 CCP22-30 under reducing (R) and nonreducing (NR) conditions. The positions of the m.w. mark- ers are indicated. (B) CD spectroscopy of CR1 CCP22–30. A spectrum was recorded in the far-UV (200–260 nm) and collected six times. The mean values for each wavelength were calculated. The maximal ellipticity is indicated by an arrowhead. (C) Electron microscopy analysis of CR1 CCP22–30 (375000) after negative staining with 2% sodium silicotungstate.

Article Snippet: Recombinant soluble human CR1 was purchased from R&D Systems Europe (Lille, France).

Techniques: SDS Page, Circular Dichroism, Electron Microscopy, Negative Staining

Journal: Journal of Biological Chemistry

Article Title: Growth Factor Induction of Cripto-1 Shedding by Glycosylphosphatidylinositol-Phospholipase D and Enhancement of Endothelial Cell Migration

doi: 10.1074/jbc.m702713200

Figure Lengend Snippet: FIGURE 1. Lipid raft localization and release of CR-1 protein. A, NTERA2/D1 cells were stained with anti-CR-1 mAb (green), and lipid raft marker GM-1 was labeled with CTxB (red) without permeabilization and analyzed by confocal microscopy (upper panel). Magnified images for the marked regions in the upper panel are shown in the lower left panel. Negative controls without pri- mary antibody are shown in the lower right panel. Nuclei were stained with DAPI (blue) in all merged images. Scale bar 10 m. B, biochemical isolation of DRMs with 1% Triton X-100 by sucrose-gradient centrifugation of NTERA2/D1 cells. The gradient fractions (lanes 4–12) were analyzed with Western blot analysis for CR-1. Transferrin receptor (TfR) was used as a control for non-raft membrane protein. The lipid raft marker GM-1 in the same sam- ples was detected with HRP-conjugated CTxB by dot-blot analysis. C, compar- ative study of cell-associated (CL) and released (CM) CR-1 with or without serum by semi-quantitative Western blot analysis (see Table 1 and supple- mental Fig. S1, A–C). A representative blot for NTERA2/D1 cells is shown. The indicated amount of recombinant human CR-1 protein was used as a standard.

Article Snippet: Reagents—Human CR-1 monoclonal antibodies (mAbs) (MAB2771 and FAB2772P) were obtained from R&D Systems (Minneapolis, MN) or developed as previously reported (B3F6) (8).

Techniques: Staining, Marker, Labeling, Confocal Microscopy, Isolation, Gradient Centrifugation, Western Blot, Control, Membrane, Dot Blot, Recombinant

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Table of Sequences for Forward and Reverse Primers for Genes Analyzed by PCR

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques:

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal, but not Cripto, is expressed in adult human islets. Expression of Nodal and Cripto mRNA and protein in isolated adult human islets are shown. A, PCR for Nodal, Cripto, and activin receptors using 0.8 μg of mRNA and specific primers (Table 1) resolved on a 2% agarose gel. HPNE and PANC-1 cells were used as controls. B, Western blot for Nodal protein in isolated human islets cultured for 24 hours before islets were harvested, washed with PBS, and frozen at −80°C. C, Western blot for Cripto in isolated human islets, with MCF7 cells as a positive control.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Expressing, Isolation, Agarose Gel Electrophoresis, Western Blot, Cell Culture, Positive Control

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal stimulates human β-cell proliferation while inhibiting α-cell proliferation. Flow cytometry analysis of human islets trypisinized to single cells after 120 hours of exposure to Cripto (250 ng/mL) and/or Nodal (10 μg/mL). Cells were stained for insulin, glucagon, and BrdU to evaluate for proliferating β- and α-cells in Cripto- and/or Nodal-treated groups compared with controls. A, Gating strategy used for flow cytometry analysis. Viable cells, cells that did not take up Live/Dead stain, were gated for insulin and glucagon positive cells (red arrow). Endocrine cells were further gated for double-positive insulin +/BrdU+ (blue arrow) or glucagon+/BrdU+ (green arrow), indicating the proliferating subset of β and α-cells. (BrdU isotype is shown.) B, Comparison of proliferating β-cells in Cripto- and/or Nodal-treated islets compared with control. A total of 3773 ± 577 β-cells were counted per treatment group per experiment (n = 5). C, Comparison of proliferating α-cells in Cripto- and/or Nodal-treated islets compared with control. A total of 5707 ± 961 α-cells were counted per treatment group per experiment (n = 4). Bars indicate mean ± SEM. *, P < .05.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Flow Cytometry, Staining, Comparison, Control

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Measurement of β-cell proliferation by immunofluorescence confirms robustness of flow cytometry proliferation assay. A, Example of control and Nodal-treated (10 μg/mL) human islets embedded in paraffin, sectioned, and stained for insulin (green), BrdU (red), and DAPI (blue). β-Cells were considered BrdU+ if BrdU and DAPI colocalized and insulin staining surrounded the nucleus (white arrow). B, Quantification of BrdU-positive β-cells in islets after 120 hours exposure to Cripto 250 ng/mL, Nodal 10 μg/ml, or Nodal (10 μg/mL) + Cripto (250 ng/mL) compared with control. Insulin+/BrdU+ cells were quantified per total number of insulin+ cells and results are shown as a percentage of control [n = 4 separate experiments (mean number of β-cells counted per group per experiment: control, 3359 ± 265; Cripto, 3074 ± 253; Nodal, 3137 ± 414; Nodal + Cripto, 3628 ± 280]. C, Quantification of Ki67-positive β-cells in islets after 120 hours exposure to Nodal 10 μg/mL compared with control. Insulin+/Ki67+ cells were quantified per total number of insulin+ cells and results are shown as a percentage of control [n = 3 separate experiments (mean number of β-cells counted per group per experiment: control, 4245 ± 73; Nodal, 4247 ± 175]. D, Example of control and Nodal-treated (10 μg/ml) human islets stained for insulin (green), Ki67 (red), and DAPI (blue). β-Cells were considered Ki67+ if Ki67 and DAPI colocalized and insulin staining surrounded the nucleus (white arrow). Bars indicate mean ± SEM. Scale bar, 50 μM. *, P < .05.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Immunofluorescence, Flow Cytometry, Proliferation Assay, Control, Staining

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal signals through SMAD proteins in adult human islets, whereas Nodal and Cripto have no effect on acute or chronic AKT or MAPK activity. Western blot analysis of cell signaling pathways in human islets exposed to acute (30 min) treatment with Nodal (10 μg/mL) and/or Cripto (250 ng/mL). A, Phosphorylated-SMAD2 (n = 5). B, Phosphorylated AKT (n = 3) and phosphorylated ERK 1/2 (n = 4). Western blot analysis of cell signaling pathways in human islets exposed to chronic (120 h) treatment with Nodal (10 μg/mL) and/or Cripto (250 ng/mL). C, Phosphorylated-AKT and ERK 1/2 (n = 3–4). Bars indicate mean ± SEM. *, P < .05.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Activity Assay, Western Blot, Protein-Protein interactions

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Chronic Nodal and Cripto treatment does not enhance dedifferentiation of human islet cells. Western blot and immunofluorescent analysis of markers of differentiation and cell signaling pathways in human islets were conducted at baseline compared with untreated cultured islets (control) and islets exposed to chronic (120 h) treatment with Nodal (10 μg/mL) and/or Cripto (250 ng/mL). A, Representative images of CK19 expression in baseline, control, and treated islets after 120 hours of culture. CK19 (red), insulin (green), and DAPI (blue) are shown. B, CK19 and SOX9 expression in baseline islets compared with control and treated islets after 120 hours culture (n = 3 for both). Bars indicate mean ± SEM.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Western Blot, Protein-Protein interactions, Cell Culture, Control, Expressing

Journal: Endocrinology

Article Title: TGF-β Superfamily Member Nodal Stimulates Human β-Cell Proliferation While Maintaining Cellular Viability

doi: 10.1210/en.2013-1197

Figure Lengend Snippet: Nodal and Cripto do not adversely affect viability or stimulate apoptosis in human islets. A, Viability of human islet cells by flow cytometry assay (Live/Dead) after 120 hours of exposure to Nodal (10 μg/mL) and/or Cripto (250 ng/mL) compared with control. Live cells are shown within the gate (n = 5). B, TUNEL staining of human islets exposed to Nodal (10 μg/mL) and Cripto (250 ng/mL) for 120 hours compared with control (n = 3). Arrows indicate apoptotic cells (as indicated by TUNEL+/DAPI+). Arrowheads indicate examples of nonspecific fluorescence (DAPI−) that were not counted as apoptotic cells. Then 49 ± 1.4 islets were counted per treatment group per experiment. Bars indicate mean ± SEM. Scale bar, 50 μM. C, Western blot for cleaved caspase-3 expression in islets cultured for 120 hours exposed to Cripto (250 ng/mL) and/or Nodal (10 μg/mL) compared with control. Human islets exposed to staurosporine (STS) 0.5 μM for 18 hours were used as positive control. Image shown is representative of three separate experiments.

Article Snippet: After 24 hours in serum-free media, recombinant human Nodal (R&D Systems) and recombinant human Cripto (R&D Systems) were added to the media.

Techniques: Flow Cytometry, Control, TUNEL Assay, Staining, Fluorescence, Western Blot, Expressing, Cell Culture, Positive Control