Journal: Biology of reproduction

Article Title: Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

doi: 10.1095/biolreprod.110.084160

Figure Lengend Snippet: FIG. 5. Signaling pathways involved in MAPK3/1 phosphorylation induced by G-1 in the Sertoli cells. Cells were incubated in the absence (C, control) and presence of G-1 (1 nM). Cells were untreated or pretreated with the SRC family of protein tyrosine kinases inhibitor (PP2, 5 nM, 30 min); A metalloprotease inhibitor GM6001 (GM200 nM, 30 min; A), MAP2K1/2 inhibitor (U0126, 20 lM, 30 min; A), pertussis toxin (PTX, 100 lM, 16 h; B), and EGFR kinase inhibitor (AG 1478, 50 lM, 15 min; B). Afterwards, cells were stimulated with G-1 (1 nM, 10 min). Total cell lysates (60 lg of protein/lane) were resolved in 10% SDS/PAGE, transferred to PVDF membrane, and probed with antibody specific for phosphorylated MAPK3/1 (top panels) or with antibody that recognizes total (phosphorylation state-independent) MAPK3/1 proteins (bottom panels). The relative positions of phosphorylated MAPK3/1 and total MAPK3/1 proteins are shown at the right. The data shown are representative of four independent experiments. Right panels: bars represent the densitometric analysis of the Western blot assays. Solid bars¼MAPK3; open bars¼MAPK1. Results were normalized to total MAPK3/1 expression in each sample and plotted (mean 6 SEM) in relation to control, C (¼1). *MAPK3/1 activation significantly greater than that control (P , 0.05, Student t-test).

Article Snippet: Cells were also untreated or pretreated with MAP2K1/2 inhibitor U0126 (20 lM; Cell Signaling) for 30 min or the EGFR kinase inhibitor AG 1478 (50 lM; Calbiochem) for 15 min at 358C [22, 43– 44].

Techniques: Protein-Protein interactions, Phospho-proteomics, Incubation, Control, SDS Page, Membrane, Western Blot, Expressing, Activation Assay

Journal: Biology of reproduction

Article Title: Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

doi: 10.1095/biolreprod.110.084160

Figure Lengend Snippet: FIG. 6. Effects of E2 and G-1 on CCND1 expression in the Sertoli cells. A) Cells were incubated in the absence (C, control) and presence of E2 (0.1 nM) or G-1 (1 nM) for 24 h. B) Cells were untreated or pretreated with EGFR kinase inhibitor (AG 1478, 50 lM, 15 min) or MAP2K1/2 inhibitor (U0126, 20 lM, 30 min). Afterwards, cells were stimulated with E2 (0.1 nM, 24 h). Total cell lysates (40 lg of protein/lane) were resolved in 15% SDS/PAGE, trans- ferred to PVDF membrane, and probed with antibody specific for CCND1. Results were normalized to ACTB expression in each sample and plotted (mean 6 SEM) in relation to control, C (¼100%). *CCND1 expression significantly different from con- trol (P , 0.05, Student t-test).

Article Snippet: Cells were also untreated or pretreated with MAP2K1/2 inhibitor U0126 (20 lM; Cell Signaling) for 30 min or the EGFR kinase inhibitor AG 1478 (50 lM; Calbiochem) for 15 min at 358C [22, 43– 44].

Techniques: Expressing, Incubation, Control, SDS Page, Membrane

Journal: Biology of reproduction

Article Title: Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

doi: 10.1095/biolreprod.110.084160

Figure Lengend Snippet: FIG. 7. Effects of G-1 on BAX and BCL2 expression in the Sertoli cells. A) Cells were incubated in the absence (C, control) and presence of G-1 (1 nM) for 24 h. B) Cells were untreated or pretreated with EGFR kinase inhibitor (AG 1478, 50 lM, 15 min) or MAP2K1/2 inhibitor (U0126, 20 lM, 30 min). Afterwards, cells were stimulated with G-1 (1 nM, 24 h). Total cell lysates (40 and 60 lg of protein/lane, respectively for BAX and BCL2) were resolved in 15% SDS/ PAGE, transferred to PVDF membrane, and probed with antibody specific for BAX and BCL2. Results were normalized to ACTB expression in each sample and plotted (mean 6 SEM) in relation to control, C (¼100%). *BAX and BCL2 expression sig- nificantly different from control (P , 0.05, Student t-test).

Article Snippet: Cells were also untreated or pretreated with MAP2K1/2 inhibitor U0126 (20 lM; Cell Signaling) for 30 min or the EGFR kinase inhibitor AG 1478 (50 lM; Calbiochem) for 15 min at 358C [22, 43– 44].

Techniques: Expressing, Incubation, Control, SDS Page, Membrane

Journal: Biology of reproduction

Article Title: Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

doi: 10.1095/biolreprod.110.084160

Figure Lengend Snippet: FIG. 8. Effects of E2 on BAX and BCL2 expression in the Sertoli cells. A) Cells were incubated in the absence (C, control) and presence of E2 (0.1 nM) for 24 h. B) Cells were untreated or pretreated with EGFR kinase inhibitor (AG 1478, 50 lM, 15 min) or MAP2K1/2 inhibitor (U0126, 20 lM, 30 min). Afterwards, cells were stimulated with E2 (0.1 nM, 24 h). C) Cells were untreated or pretreated with ICI 182,780 (1 nM, 30 min). Afterwards, cells were stimulated with E2 (0.1 nM, 24 h). Total cell lysates (40 and 60 lg of protein/lane, respectively, for BAX and BCL2) were resolved in 15% SDS/ PAGE, transferred to PVDF membrane, and probed with antibody specific for BAX and BCL2. Results were normalized to ACTB expression in each sample and plotted (mean 6 SEM) in relation to control, C (¼100%). *BAX and BCL2 expression sig- nificantly different from control (P , 0.05, Student t-test).

Article Snippet: Cells were also untreated or pretreated with MAP2K1/2 inhibitor U0126 (20 lM; Cell Signaling) for 30 min or the EGFR kinase inhibitor AG 1478 (50 lM; Calbiochem) for 15 min at 358C [22, 43– 44].

Techniques: Expressing, Incubation, Control, SDS Page, Membrane

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: 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 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