Journal: Frontiers in Genetics

Article Title: A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

doi: 10.3389/fgene.2018.00498

Figure Lengend Snippet: List of genes from expressed in the different stages of differentiation and maturation of SCs based on literature search.

Article Snippet: Human foreskin fibroblast (HFFn, PC501 A-HFF, System Biosciences Mountain View, CA, United States) were cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep according to the manufacturer instructions.

Techniques: Wilms Tumor Assay, Binding Assay

Journal: Frontiers in Genetics

Article Title: A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

doi: 10.3389/fgene.2018.00498

Figure Lengend Snippet: Graphic representation of Human Fibroblasts (HFs) cell reprogramming and iPSCs differentiation toward SLCs. AP, alkaline phosphatase; EBs, embrioid bodies; FGF9, fibroblast growth factor 9; IF, immunofluorescence; iPSCs, induced pluripotent stem cells; PGD2, prostaglandin 2; RNA-seq, next generation RNA sequencing; SLCs, Sertoli-like cells; SNL, SNL feeder cells.

Article Snippet: Human foreskin fibroblast (HFFn, PC501 A-HFF, System Biosciences Mountain View, CA, United States) were cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep according to the manufacturer instructions.

Techniques: Immunofluorescence, RNA Sequencing

Journal: Frontiers in Genetics

Article Title: A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

doi: 10.3389/fgene.2018.00498

Figure Lengend Snippet: (A) Immunofluorescence staining of iPSC colonies. Zoom-in details in right-down squares. Cells were stained with antibodies against OCT4, TRA-1-81, SSEA3, SSEA4, or SSEA1 and SOX2. Mounting medium contains DAPI stain. Pluripotency markers OCT4, TRA-1-81, and SOX2 positively marked the nuclei of cells. SSEA3 and SSEA4 were localized in the cytoplasmic area of colonies. SSEA1 expression was absent in colony nuclei but observed in the feeder cells. (B) RT-PCR analysis of pluripotential colonies for the genes DPPA2, DPPA4, ESG1, SOX2, and NANOG. Primers used for SOX2 and NANOG specifically detect transcripts from the endogenous genes, but not from the lentiviral transgenes. Four (1–4) different colonies are shown. HF, terminally differentiated fibroblasts; iPSC, induced pluripotent stem cells. (C,D) Teratoma formation analysis. Histology sections of teratoma stained with Hematoxylin & eosin. 1: mesoderm, 2: endoderm, 3: ectoderm. Scale bars 50 μm for (A,C) , and 100 μm for (D) .

Article Snippet: Human foreskin fibroblast (HFFn, PC501 A-HFF, System Biosciences Mountain View, CA, United States) were cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep according to the manufacturer instructions.

Techniques: Immunofluorescence, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Genetics

Article Title: A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

doi: 10.3389/fgene.2018.00498

Figure Lengend Snippet: Immunofluorescence staining of SLC colonies with antibodies against SOX9. Mounting medium contains DAPI stain. SOX9 were expressed in SLC colonies. HF, terminally differentiated fibroblasts; HSerCs, human Sertoli cells; NT2, NT2d1 cells; SOX9, SRY (sex determining region Y)-box 9. Scale bars 50 μm.

Article Snippet: Human foreskin fibroblast (HFFn, PC501 A-HFF, System Biosciences Mountain View, CA, United States) were cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep according to the manufacturer instructions.

Techniques: Immunofluorescence, Staining

Journal: Frontiers in Genetics

Article Title: A Human Gonadal Cell Model From Induced Pluripotent Stem Cells

doi: 10.3389/fgene.2018.00498

Figure Lengend Snippet: Secreted AMH quantification by ELISA. SLCs secreted AMH levels were significantly higher when compared with AMH secretion by HSerCs or NT2d1 cells. HF: terminally differentiated fibroblasts, HSerCs: primary human Sertoli cells, NT2d1: NT2d1 cells, SLCs: Sertoli like cells. n = 4 for SLCs and HSerCs, n = 2 for HFs and NT2d1. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001.

Article Snippet: Human foreskin fibroblast (HFFn, PC501 A-HFF, System Biosciences Mountain View, CA, United States) were cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep according to the manufacturer instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Cell Biology

Article Title: Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

doi:

Figure Lengend Snippet: Coexpression of either myr-PKCε, V38R-Ras, L61Rac1, or p110α-CAAX with tac-β1 can restore cell spreading on collagen I. (A) Diagram of the control tac receptor containing the extracellular and transmembrane domains of the small (tac) subunit of the human interleukin-2 receptor and the tac-β1 chimera containing the same domains of the interleukin-2 receptor fused to the human integrin β1A cytoplasmic domain. (B) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myr-PKCε-Flag (c and e) or tac-β1 and myc-V38R-Ras (d and f). Cell area for 100 randomly sampled positively transfected cells is plotted as a function of either tac epitope expression (a–d), myr-PKCε-Flag expression (e) for the same cells shown in c, or myc-V38R-Ras expression (f) for the same cells shown in d. The x axis is a linear scale of cell area from 0 to 1,600 μm 2 , the y axis is a linear scale of either FITC fluorescence (tac epitope expression) units defined by Image Pro-Plus from 0 to 2.4 × 10 4 (a–d) or rhodamine fluorescence (Flag or myc epitope expression) from 0 to 1.2 × 10 5 (e and f). (C) Fibroblasts were transfected with tac (a) or tac-β1 (b), or cotransfected with tac-β1 and myc-L61Rac1 (c and d). Cell area for 90 randomly sampled cells expressing tac, tac-β1, or coexpressing tac-β1 and myc-L61Rac1 is plotted as a function of tac epitope expression (a–c) or myc epitope expression (d) for the cells shown in c. The x axis is a linear scale of cell area from 0 to 2,400 μm 2 , the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 2.4 × 10 4 (a–c) or rhodamine fluorescence (myc epitope expression) from 0 to 1.2 × 10 5 (d). (D) Fibroblasts transfected with the control tac receptor (a) or tac-β1 (b) or tac-β1 and p110α-CAAX (c) were analyzed for cell-surface expression of the tac epitope and cell area, as described in Materials and Methods. Cell area for 98 randomly sampled positively transfected cells is plotted as a function of tac epitope expression. The x axis is a linear scale of cell area from 0 to 2,400 μm 2 and the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 3.2 × 10 4 (a) or 0 to 1.6 × 10 4 (b and c). (B–D) The vertical line positioned at a cell area of 560 μm 2 indicates the separation of spread (right) and not spread (left) cells. It is important to note that our spreading assays primarily analyze cells expressing moderate to low levels of tac-β1, since cell attachment to collagen I is inhibited by the expression of high levels of tac-β1 (data not shown). This observation is consistent with our recent studies showing that high levels of tac-β1 inhibit cell attachment to fibronectin . The range of FITC fluorescence represents the range of tac-β1 expression detected in the adherent transfected cells. These experiments were performed three times and similar results were obtained.

Article Snippet: Primary human foreskin fibroblasts (Vec Technologies) were maintained in DMEM supplemented with 10% fetal bovine serum and 2 mM l -glutamine.

Techniques: Control, Transfection, Expressing, Fluorescence, Cell Attachment Assay

Journal: The Journal of Cell Biology

Article Title: Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

doi:

Figure Lengend Snippet: PI 3-kinase activity is required for V38R-Ras, but not for L61Rac1 or myr-PKCε, to rescue cell spreading. Normal fibroblasts were transfected with either tac alone (A, a and e, and B, a and c) or cotransfected with tac-β1 and either V38R-Ras (B, b and d), p110α-CAAX (A, b and f), L61Rac1 (A, c and g), or myr-PKCε (A, d and h), and the morphology of cells adherent to collagen was analyzed in the presence of the PI 3-kinase inhibitor, LY294002 (A, e–h, and B, c and d), or DMSO (A, a–d, and B, c and d) as described in Materials and Methods. In each case, 100 (A) or 95 (B) randomly sampled transfected cells were analyzed for tac epitope expression and cell area as described previously. The x axis is a linear scale of cell area from 0 to 2.4 × 10 3 μm 2 (A) or 0 to 3.2 × 10 3 μm 2 (B), and the y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 4.8 × 10 4 (A, a and e) or 0 to 2.4 × 10 4 (A, b–d and f–h, and B, b, d) or 0 to 8.0 × 10 4 (B, a and c). The vertical line positioned at 560 μm 2 indicates the separation of spread (right) and not spread (left) cells. These experiments were performed three times and similar results were obtained.

Article Snippet: Primary human foreskin fibroblasts (Vec Technologies) were maintained in DMEM supplemented with 10% fetal bovine serum and 2 mM l -glutamine.

Techniques: Activity Assay, Transfection, Expressing, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

doi:

Figure Lengend Snippet: The morphology of cotransfected cells adherent to collagen I. (A) The morphology of cells expressing tac or tac-β1 alone or cells coexpressing tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε. Shown is tac epitope expression (FITC fluorescence) for representative cells from the quantitative experiment shown in . Also included is tac-β1 expression of a representative cell from the cotransfection of tac-β1 and p110α-CAAX. Fluorescence images were obtained with Spot software and composites were generated in adobe photoshop. Scale bar: 10 μm. (B) Coexpression of either V38R-Ras, L61Rac1, or myr-PKCε in addition to rescuing cell spreading also restores the localization of tac-β1 to the focal contact. Fibroblasts were cotransfected with tac-β1 and either V38R-Ras, L61Rac1, or myr-PKCε, and cells adherent to collagen were costained for tac and signaling protein expression as described previously. The tac-FITC staining (right) and corresponding interference reflection pattern (left) of coexpressing cells is shown. Scale bar: 10 μm.

Article Snippet: Primary human foreskin fibroblasts (Vec Technologies) were maintained in DMEM supplemented with 10% fetal bovine serum and 2 mM l -glutamine.

Techniques: Expressing, Fluorescence, Cotransfection, Software, Generated, Staining

Journal: The Journal of Cell Biology

Article Title: Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

doi:

Figure Lengend Snippet: The morphology of cells expressing either p110α-CAAX, L61Rac1, N17Rac1, V38R-Ras, N43R-Ras, or myr-PKCε. Normal fibroblasts were transfected with tac alone or each of the signaling proteins, and subsequently replated onto collagen I as described in Materials and Methods. Adherent cells were immunostained for expression of the tac epitope in cells transfected with the control tac receptor alone or cotransfected with the control tac receptor and p110α-CAAX. Cells were immunostained for expression of the flag epitope in cells transfected with myr-PKCε-Flag, or the myc epitope in the case of myc-L61Rac1, myc-N17Rac1, myc-V38R-Ras, and myc-N43R-Ras. Staining was visualized using rhodamine-conjugated secondary antibodies. Fluorescence images were obtained as described in . Scale bar: 10 μm.

Article Snippet: Primary human foreskin fibroblasts (Vec Technologies) were maintained in DMEM supplemented with 10% fetal bovine serum and 2 mM l -glutamine.

Techniques: Expressing, Transfection, Control, FLAG-tag, Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

doi:

Figure Lengend Snippet: The rescue of tac-β1–inhibited cell spreading on collagen I is a β1 integrin–dependent process. Human fibroblasts transfected with the control tac receptor alone (A, a and e, and B, a and c) or cotransfected with tac-β1 and either p110α-CAAX (A, b and f), myr-PKCε (A, c and g), V38R-Ras (A, d and h), or L61Rac1 (B, b and d) were analyzed for spreading on collagen I in the presence of control ascites (A, a–d, and B, a and b) or β1 function blocking P4C10 ascites (A, e–h, and B, c and d) as described in Materials and Methods. The extent of cell spreading and the expression levels of the tac epitope were quantified for 100 (A) or 80 (B) randomly sampled positively transfected cells, and the results are shown by dot plot. The x axis is a linear scale of cell area from 0 to 3.2 × 10 3 μm 2 (A and B). The y axis is a linear scale of FITC fluorescence (tac epitope expression) from 0 to 2.4 × 10 4 (A and B). The vertical line positioned at a cell area of 560 μm 2 indicates the separation of spread (right) and not spread (left) cells. These experiments were performed twice and similar results were obtained.

Article Snippet: Primary human foreskin fibroblasts (Vec Technologies) were maintained in DMEM supplemented with 10% fetal bovine serum and 2 mM l -glutamine.

Techniques: Transfection, Control, Blocking Assay, Expressing, Fluorescence