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: 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: Cell proliferation

Article Title: Administration of Cripto in GRP78 overexpressed human MSCs enhances stem cell viability and angiogenesis during human MSC transplantation therapy.

doi: 10.1111/cpr.12463

Figure Lengend Snippet: FIGURE 2 Binding of GRP78 with Cripto enhances cellular proliferation via JAK2-STAT3 pathway. (A) Western blot analysis of p-JAK2 and p-STAT3 expressions in hMSCs, GRP78-hMSC and hMSC pre-treated with antibody GRP78 (Ab-GRP78) exposed to Cripto for 0, 5, 10 and 20 minutes. The down panel represents the phosphorylation levels of JAK2 and STAT-3 in control to β-actin. Values represent the mean ± SEM. **P < .01 vs. control, ##P < .01 vs. GRP78-hMSC. (B) hMSC, GRP78-hMSC, and Ab-GRP78 were treated with or without Cripto for 24 hour. Images of cellular proliferation measurement were shown using MTT assay. Values represent the mean ± SEM. *P < .05 vs control, **P < .01 vs. control, ##P < .01 vs treated Cripto, and $$P < .01 vs Cripto+GRP78-hMSCs. (C) Image of single cell assay after 10 days in 96 well plate, stained with Giemsa stain. Scale bar = 100 μM. (D) The number of cells per field of view in each well of a 96 well plates is plotted (n = 3). Values represent the mean ± SEM. **P < .01 vs control, ##P < .01 vs treated Cripto, and $$P < .01 vs Cripto+GRP78-hMSCs. (E) Western blot analysis of CDK 2, Cyclin E, CDK 4, and Cyclin D1 in hMSC, GRP78-hMSC and Ab-GRP78 were treated with or without Cripto for 24 hour. Values represent the mean ± SEM. *P < .05 vs control, **P < .01 vs control, #P < .05 vs treated Cripto, ##P < .01 vs treated Cripto and $$P < .01 vs Cripto+GRP78-hMSCs. (F) hMSC, GRP78-hMSC and Ab-GRP78 were treated with or without Cripto for 24 hour. Measurement of CDK4/cyclin D1 kinase activation concentration in hMSCs using ELISA (n = 3). Values represent the mean ± SEM. **P < .01 vs untreated hMSCs. ##P < .01 vs treated Cripto, and $$P < .01 vs Cripto+GRP78-hMSCs. (G) Images of flow cytometric analysis for PI staining to assess S phase populations in hMSC, GRP78-hMSC and Ab-GRP78 treated with Cripto. **P < .01 vs untreated hMSCs. ##P < 0.01 vs treated Cripto and $$P < .01 vs Cripto+GRP78-hMSCs

Article Snippet: After washing with Tris- buffered saline/Tween- 20 buffer (0.05% Tween- 20, 150 mM NaCl, 10 mM Tris- HCl; pH 7.6), membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated with primary antibodies against 78 kDa glucoseregulated protein (GRP78), SRY (sex determining region Y)- box 2 (SOX2), Nanog, octamer- binding transcription factor 4 (OCT4), phosphorylated Janus kinase 2 (p- JAK2), phosphorylated Signal transducer and activator of transcription 3 (p- STAT3), Cyclindependent kinase 2 (CDK 2), Cyclin E, Cyclin- dependent kinase 4 (CDK 4), Cyclin D1, Cripto, B- cell lymphoma 2 (BCL- 2), Bcl- 2- associated X protein (BAX), cleaved caspase- 3, cleaved Poly ADPribose polymerase 1 (c- PARP- 1), β- actin and α- tubulin (all from Santa Cruz Biotechnology).

Techniques: Binding Assay, Western Blot, Phospho-proteomics, Control, MTT Assay, Staining, Giemsa Stain, Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

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: 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 μm. 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 . (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 μm. (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, Immunofluorescence

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: CRIPTO (CR-1) expression regulates cells proliferation and stem cell gene expression in NSCLC spheroids. (A) Immunoblot analysis of CRIPTO and βActin 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: NSCLC subpopulations expressing high or low CRIPTO (CR-1) levels are interconvertible in vitro and in vivo . (A) FACS-based separation of CRIPTO high and CRIPTO low subpopulations of AC1 spheroid cultures. (B) Self-renewal capacity of sorted CRIPTO high and CRIPTO low AC1 spheroid cells, evaluated as colony formation in semisolid culture and expressed in bottom panels as normalized colony size/percentage over plated cells. (C) qRT-PCR analysis of CRIPTO and stem cell related genes (OCT3/4, SOX2, NANOG) performed on CRIPTO high and CRIPTO low subpopulations of AC1 spheroids. (D) Proliferation curve of CRIPTO high (dark pink square) and CRIPTO low (light blue dot) subpopulations or Bulk (burgundy triangle) cultures of AC1 spheroids, starting at day 0 (after sorting) and monitored at the indicated times. (E) qRT-PCR analysis of CRIPTO high (light purple histogram) and CRIPTO low (light blue histogram) subpopulations at the indicated times starting at day 0 after sorting. (F) Left: In vivo growth of tumor xenografts obtained by subcutaneous inoculation of CRIPTO high (dark pink square) and CRIPTO low (light blue dot) subpopulations or Bulk (burgundy triangle) cultures of AC1 spheroids, monitored at the indicated times. Results shown are the mean ± SEM of values obtained using 4 mice per group. *P < 0.05; **P < 0.01, ***P < 0.001. Middle: representative confocal images of CRIPTO (red) staining of CRIPTO high , CRIPTO low and Bulk tumors. 40X magnification, scale bar 50 μm. Right: quantification of CRIPTO performed on 3 sets composed of 5 fields/group. **P < 0.01. AU, arbitrary units.

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, Quantitative RT-PCR, Staining

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: 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 μM plus Gemcitabine 25 μM 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 μm. 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