Journal: Stem cells (Dayton, Ohio)

Article Title: Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed.

doi: 10.1634/stemcells.2006-0219

Figure Lengend Snippet: Figure 1. hESCs differentiate when phosphatidylinositol 3-kinase (PI3K) signaling is inhibited. (A): Collagenase/trypsin-passaged BG01 hESCs were plated on Matrigel in MEF-CM and Fgf2 and grown in the presence or absence of LY 294002 for 4 days. Bright-field images were taken at the indicated magnifications. (B): CD9 expression assayed by flow cytometry. Untreated hESCs and LY 294002-treated cells (4 days) were stained with a fluorescein isothiocyanate-conjugated anti-CD9 antibody (control, isotype-matched control antibody). (C): Immunocytochemistry of paraformal- dehyde-fixed cells. Left panel, untreated BG01 hESCs grown in MEF-CM, Fgf2, and 20% KSR; middle panel, differentiated hESCs grown for 4 days in the absence of MEF-CM and Fgf2; right panel, hESCs grown with MEF-CM, Fgf2, and 20% KSR and treated with LY 294002 for 4 days. Cells were stained as indicated with DAPI and probed simultaneously with anti-CD9 and antiSOX17 antibodies. Magnification, 20 objective. (D): Immunoblot analysis (20 g of total protein per track) of T, CDK2, and OCT4 protein levels in untreated hESCs () or during differentiation of LY 294002-treated hESCs (BG01) over 6 days. (E): Q-PCR analysis of marker transcripts associated with formation of the three germ layers (ectoderm, mesoderm, and endoderm), extraembryonic endoderm, hESCs, and mesendoderm stages of differentiation following treatment with LY 294002 (50 M) for up to 5 days. Experiments were performed in quadruplicate and expressed as the fold increase over untreated hESCs SEM. (F): Temporal changes in gene expression associated with the generation of hESC-DE (A) (supplemental online Fig. 2) paralleled that seen in early embryonic development. Abbreviations: CDK, cyclin-dependent kinase; CM, conditioned medium; d, day(s); DAPI, 4,6-diamidino-2-phenylindole; DE, definitive endoderm; Fgf, fibroblast growth factor; GSC, goosecoid; hESC, human ESC; PS, primitive streak stage of development when mesendoderm was formed.

Article Snippet: MEF-CM was produced by conditioning of mitomycin C-treated MEFs (Specialty Media, http://www.specialtymedia.com) for 24–36 hours in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/F12), high (20%) Knockout Serum Replacement (KSR) (Gibco, Grand Island, NY, http://www.invitrogen.com), 8 ng/ml human recombinant fibroblast growth factor 2 (Fgf2) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), 1 mM nonessential amino acids, L-glutamine, penicillin/streptomycin, 0.1 mM 2-mercaptoethanol.

Techniques: Expressing, Flow Cytometry, Staining, Control, Immunocytochemistry, Western Blot, Marker, Gene Expression

Journal: Stem cells (Dayton, Ohio)

Article Title: Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed.

doi: 10.1634/stemcells.2006-0219

Figure Lengend Snippet: Figure 5. Activin/Nodal specifies definitive endoderm (DE) when phosphatidylinositol-3-kinase signaling is reduced. (A): Activin-like activities work in collaboration with LY 294002 to drive formation of DE. LY 294002-dependent SOX17 expression is suppressed by inhib- itors of Activin signaling but not bone morphogenetic protein signaling or Cripto-dependent signaling. SOX17 expression was evaluated by quantitative polymerase chain reaction (Q-PCR) 4 days after addition of LY 294002 (50 M) to BG01 hESCs grown in MEF-CM, 8 ng/ml Fgf2, high (20%) KSR in the presence or absence of Chordin (500 ng/ml), Follistatin (500 ng/ml), Lefty-A (500 ng/ml), Noggin (500 ng/ml), or SB-43152 (10 M). Assays were performed in triplicate and are shown SEM. (B): MEF-CM can be substituted by recombinant Activin A or Nodal to promote LY 294002-dependent DE formation. SOX17 mRNA levels (fold increase over untreated) were evaluated by Q-PCR under various conditions over 4 days; shown are LY 294002 (50 M), Activin A (100 ng/ml), and Nodal (1 g/ml). UCM was composed of Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium supple- mented with 4 ng/ml Fgf2, high (20%) KSR. (C): Changes in AKT1 activity under different DE differentiation conditions. hESCs were grown in unconditioned medium in low (2%) KSR plus 100 ng/ml Activin A or in MEF-CM containing high (20%) KSR and LY 294002 (50 M). AKT1 activity was evaluated by probing cell lysates (20 g of protein) using an activity-specific antibody directed against serine 473 (S473). Abbreviations: d, day(s); hESC, human ESC; KSR, knockout serum replacement; MEF-CM, mouse embryo fibroblast-conditioned medium; UCM, unconditioned medium.

Article Snippet: MEF-CM was produced by conditioning of mitomycin C-treated MEFs (Specialty Media, http://www.specialtymedia.com) for 24–36 hours in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DMEM/F12), high (20%) Knockout Serum Replacement (KSR) (Gibco, Grand Island, NY, http://www.invitrogen.com), 8 ng/ml human recombinant fibroblast growth factor 2 (Fgf2) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), 1 mM nonessential amino acids, L-glutamine, penicillin/streptomycin, 0.1 mM 2-mercaptoethanol.

Techniques: Expressing, Inhibition, Real-time Polymerase Chain Reaction, Recombinant, Modification, Activity Assay, Knock-Out

Journal: bioRxiv

Article Title: Multiple FGFR1 mutations modulate tumorigenic mechanisms in glioneuronal tumors

doi: 10.1101/2025.05.27.654799

Figure Lengend Snippet: R661P reduces K656E and N546K mutant protein stability by rescuing lysosomal degradation. A) Receptor stability experimental design: HEK293 cells expressing WT and mutant FGFR1 are stimulated with human FGF2 (hFGF2, 25 ng/mL) to induce receptor internalization and degradation and, in parallel, with cycloheximide (100 µg/mL) to block protein synthesis. B and D) Western blot analysis of a representative time-course experiment comparing WT FGFR1, K656E (B) and N546K (D) single and double mutants showing total Flag-FGFR1 protein levels at 0, 3 and 6 hours post-treatment with FGF2 and cycloheximide (CHX). C and E) Relative amounts of WT, single and double mutant FGFR1 protein obtained by quantifying western blots from n = 3 independent experiments. Values have been normalized (FC) against each relative 0h reference values. Data is represented by mean ± SEM and significant variations in protein amounts against each specific reference values have been indicated (ANOVA test, p value * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001) in the bar plots.

Article Snippet: Next, cells were induced with 25 ng/mL of recombinant human FGF2 (RD Systems) and treated with 100 µg/mL of Cycloheximide (Merck) and 200 nM of Bafilomycin A1 (Merck).

Techniques: Mutagenesis, Expressing, Blocking Assay, Western Blot