Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) expression increased in human umbilical artery endothelial cells (HUAECs), human umbilical vein endothelial cells (HUVECs), and rat brain microvascular endothelial cells (RBMECs) stimulated with VEGF. (A,B) Endothelial cell (EC) migration distance was evaluated by scratch assay. ECs were treated with a dose curve of RGMa (500–3000 ng/ml), and images were taken at the beginning and 12 h. (C,D) ECs were treated with RGMa (2 µg/ml), and lysates were collected over a time course of 50 min. The amount of phosphorylated focal adhesion kinase (p-FAK) protein was visualized with western blot analysis. (E,F) ECs were treated with VEGF (50 ng/ml), and lysates were collected over a course of 120 min. p-FAK protein levels were visualized with western blot analysis. (G–I) Quantitative real-time polymerase chain reaction showed RGMa mRNA level was upregulated in HUAECs, HUVECs, and RBMECs exposed to VEGF (50 ng/ml) for 30 min. (J–L) RGMa protein levels were visualized in HUAECs, HUVECs, and RBMECs exposed to VEGF at 30 min by western blot analysis. Data in bar graphs represent the means ± SD of ≥4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, # VS 1500 ng/ml, ### P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Migration, Wound Healing Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) suppressed VEGF expression, phosphorylation of focal adhesion kinase (FAK), proliferation, migration, and tube formation in ECs. (A,B) Lysate was collected, and VEGF was detected by western blot in human umbilical artery endothelial cells (HUAECs) treated with RGMa (2 µg/ml); (C–E) ELISA kit assay showed VEGFA decreased in endothelial cell (EC)-culture supernatant exposed to RGMa compared with cell-culture supernatant from control group. (F–H) Cell proliferation was evaluated with cell-counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays (I,J) . RGMa decreased proliferation of ECs stimulated and unstimulated with VEGF. (K,L) FAK (Tyr397) phosphorylation was measured with western blot in HUAECs treated with vehicle, RGMa (2 µg/ml), VEGF (50 ng/ml), or VEGF plus RGMa. (M–P) ECs were grown to 100% confluence, serum-starved overnight, wounded with a sterile pipette tip to remove cells, and treated with control, RGMa, VEGF, or VEGF plus RGMa. Photographs (40×) were taken at 12 h after injury. Wound closure of ≥3 wells was quantified and reported as mean ± SD. (Q–T) Migration activity of ECs treated with RGMa, VEGF, VEGF plus RGMa, or control was measured with transwell assay. Photographs (200×) were taken 18 h after treatment. (U–X) HUAECs were starved overnight, treated as indicated, and seeded into 96-well plates coated with Matrigel. Photographs (40×) were taken at 3 h after treatment. The number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Phospho-proteomics, Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Cell Counting, Sterility, Transferring, Activity Assay, Transwell Assay

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited angiogenesis in vitro via neogenin. (A) Human umbilical artery endothelial cells (HUAECs) were transfected with CRISPR/Cas9 neogenin knockout kit and purified with puromycin, then the result of neogenin knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or neogenin gRNA were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa. The focal adhesion kinase (FAK) (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or neogenin gRNA were determined by scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: In Vitro, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Phospho-proteomics, Migration

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Unc5b is involved in the effect of repulsive guidance molecule a (RGMa) on phosphorylation of focal adhesion kinase (FAK), migration, and tube formation in human umbilical artery endothelial cells (HUAECs). (A) HUAECs were transfected with CRISPR/Cas9 Unc5b knockout kits and purified with puromycin, then the effect of Unc5b knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or Unc5b gRNA were treated with control, RGMa, VEGF, or VEGF plus RGMa. FAK (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or Unc5b gRNA were determined with scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length was analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Phospho-proteomics, Migration, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Control

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited cytoskeleton reassembly, filopodia, and lamellipodia formation in human umbilical artery endothelial cells (HUAECs) via neogenin and Unc5b. Before the immunofluorescence experiment, HUAECs were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa for 40 min. F-actin was stained with phalloidin conjugated with FITC and phosphorylated focal adhesion kinase (p-FAK) connected with primary antibody was labeled with Alexa Fluor 555 donkey anti-rabbit (H + L) secondary antibody. (A) Immunofluorescence showed the cytoskeleton (green) change and p-FAK (red) distribution. (B) Immunofluorescence showing the cytoskeleton change of HUAECs transfected with SgRNA or neogenin gRNA. (C) Immunofluorescence showed the cytoskeleton change of HUAECs transfected with SgRNA or Unc5b gRNA. The filopodia are indicated as sharp spikes (arrowhead), and lamellipodia (arrow) are indicated as flat intensive staining. The merged images are shown in the upper panels, and the amplified indicated areas are shown in the lower panel for different groups. Photographs were obtained with laser scanning confocal microscopy (Nikon, A1 + R, magnification 400×). Results shown are representative images of ≥4 independent experiments.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Immunofluorescence, Staining, Labeling, Transfection, Amplification, Confocal Microscopy

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 1. IL-17F induces IL-8 production by NHEKs. (a) IL-8 gene expression by IL-17F in NHEKs. Total RNA was extracted from the cell lysates 4 and 24 hours after stimulation with 100 ng ml1 IL-17F, 100 ng ml1 TNF-a, 100 ng ml1 IL-17A, and PBS control. The levels of mRNA for IL-8 were calculated as the fold induction compared with the PBS control using real- time PCR. (b) Analysis of IL-8 production in NHEKs stimulated with IL-17F (100 ng ml1), TNF-a (100 ng ml1), IL-17A (100 ng ml1), and PBS control. IL-8 protein release in the supernatant was determined by ELISA as described in ‘‘Materials and Methods’’. Results represent mean±SEM from at least three independent experiments. *Po0.05 was considered significant.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 2. IL-8 gene expression in the mouse skin induced by IL-17F. Ear tissue from the IL-17F-treated and PBS control mice were collected 24 hours following intradermal injection and total RNA was isolated from each ear specimen. The primer sequence was as follows: upstream 50-ATGGCTGGG ATTCACCTCAA-30, downstream 50-AAGCCTCGCGACCATTCTT-30. The expression of IL-8 mRNA 24 hours was 2.9-fold higher than that in the control group. Results represent mean±SEM from three independent experiments.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Gene Expression, Control, Injection, Isolation, Sequencing, Expressing

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 3. Effect of inhibitors on IL-8 protein production in NHEKs. The cells were preincubated with varying concentrations of PD98059, U0126, SB202190, SP600125, or Me2SO vehicle for 1 hour, followed by stimulation with IL-17F (100 ng ml1) for 24 hours. Results represent mean±SEM from three independent experiments. *Po0.05 was considered significant versus IL-17F-stimulated cells without addition of inhibitors.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques:

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 4. Treatment with IL-17F induces ERK1/2 phosphorylation in the mouse skin. Ear tissue samples collected at 0.5, 1, and 2 hours following IL-17F injection (100 ng in 50 ml of PBS) were prepared. Western blot analysis of phosphorylated and total ERK1/2 was performed on these skin samples. The activation of ERK1/2 was seen at 0.5, 1, and 2 hours after injection. The results shown are representative of three separate experiments.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Phospho-proteomics, Injection, Western Blot, Activation Assay

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 5. Histologic examination. Histopathological findings 48 hours after intradermal injection of (a) IL-17F, (c) TNF-a, (d) IL-17A, and (e) PBS control. Bar ¼ 100 mm. (b) High magnification of a. Infiltrating cells in the dermis consisted mainly of neutrophils (arrow). Bar ¼ 50 mm. (g) A cell count was performed as described in ‘‘Materials and Methods’’, and the density of dermal infiltration was expressed as mononuclear cells, neutrophils, eosinophils, and total number of cells. No infiltrating eosinophils were seen in both the IL-17F-treated and other groups. The number of neutrophils in the dermis was significantly increased by injection of recombinant mouse IL-17F compared with the control group (*Po0.05) and the inflammatory cells in the dermis were significantly but not completely blocked by anti-IL-8 antibody (*Po0.05) (f, g). Bar ¼ 100 mm.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Injection, Control, Cell Counting, Recombinant

Journal: The Journal of investigative dermatology

Article Title: Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

doi: 10.1038/jid.2008.294

Figure Lengend Snippet: Figure 6. IL-17F protein in the skin of psoriasis patients. (a) Representative results from analysis of IL-17F protein production in the nonlesional and lesional skin of psoriasis patients. Skin specimens were homogenized and the supernatants were examined by western blotting. (b) Protein level of IL-17F in the psoriatic lesional skin was higher than in nonlesional psoriatic skin (*Po0.05). Low level of IL-17F protein was also detected in nonlesional psoriatic skin. The results were expressed as the mean±SEM (n ¼ 5). *Po0.05 was considered significant.

Article Snippet: In some experiments described below, rmTNF-a (100 ng in 50 ml of PBS), rm IL-17A (100 ng in 50 ml of PBS), rm IL-17F plus anti-human IL-8 antibody (100 ng rm IL-17F and 100 ng antihuman IL-8 Ab in total 50 ml of PBS) (all R&D Systems) were also intradermally administrated.

Techniques: Western Blot

Journal: PLoS Pathogens

Article Title: TIM-3 Does Not Act as a Receptor for Galectin-9

doi: 10.1371/journal.ppat.1003253

Figure Lengend Snippet: A) Characterization of T cell stimulator cells. Upper and middle panel: Expression of membrane-bound anti-CD3 antibody fragments (mb-aCD3) on T cell stimulator cells was detected with Dylight-649-labelled goat-anti-mouse IgG (H+L-chain specific) antibodies (grey histograms). Open histogram: reactivity with control Bw cells. Lower panel: T cell stimulator cells engineered to express human galectin-9 were probed with galectin 9 antibodies (grey histogram) or isotype control antibodies (open histogram). B) Human T cells derived from healthy donors were co-cultured with T cell stimulator cells expressing human galectin-9 or no human costimulatory molecule (control). 3 [H]-methyl-thymidine uptake was assessed following 3 days of co-culture (cpm, counts per minute). The results summarize 16 experiments with different donors. C) Culture supernatant derived from T cells activated with galectin-9 expressing stimulator cells or control stimulator cells was harvested after 48 h and subjected to multiplex cytokine measurement. Data show +/− SD of triplicates from one experiment representative for 16 independently performed.

Article Snippet: Mouse and human TIM-3 immunoglobulin (Ig) fusion proteins, human ILT5 Ig, human TROP2 Ig, human TREML2 Ig, mouse CTLA4 Ig fusion proteins - all expressed in NSO-cells – and recombinant human (rh) and recombinant mouse (rm) galectin-9 were purchased from R&D systems.

Techniques: Expressing, Membrane, Derivative Assay, Cell Culture, Co-Culture Assay, Multiplex Assay

Journal: PLoS Pathogens

Article Title: TIM-3 Does Not Act as a Receptor for Galectin-9

doi: 10.1371/journal.ppat.1003253

Figure Lengend Snippet: A, D) Bw control cells (open histograms) or Bw cells transduced to express human (A) or murine (D) galectin-9 (grey histograms) were probed with galectin-9 antibodies or with immunoglobulin fusion proteins representing human TIM-3 (TIM3-Ig; A) or murine TIM-3 (m-TIM-3-Ig; D) or a control fusion protein (B7-H3-Ig). Bound antibodies were detected with PE-labelled donkey-anti-goat antibodies. Immunoglobulin fusion proteins were detected with PE-labelled goat antibodies to the Fc-part of human IgG. B, E) Bw control cells (open histogram) or Bw cells transduced to express human (B) or murine (E) TIM-3 (grey histograms) were probed with appropriate anti-TIM-3 mAbs or with biotin-labelled recombinant human (B) or murine (E) galectin-9. Bound anti-human TIM-3 mAb and anti-murine TIM-3 mAb were detected with PE-labelled goat-anti-mouse IgG and APC-labelled goat-anti-rat IgG, respectively. SA-PE was used as secondary reagent for biotin-labelled recombinant human and murine galectin-9. C, F) Recombinant human (C) or murine (F) galectin-9 was immobilized on ELISA plates and probed with immunoglobulin fusion proteins (Ig) representing the extra-cellular domain of human TIM-3 (TIM-3-Ig; C) or murine TIM-3 (m-TIM-3-Ig; F) at the indicated concentrations. HRP-conjugated goat-anti-human IgG-Fc-specific antibodies were used for detection. Binding buffer only (PBS) or the indicated immunoglobulin fusion proteins were used as controls. All binding experiments were repeated three times with similar outcome.

Article Snippet: Mouse and human TIM-3 immunoglobulin (Ig) fusion proteins, human ILT5 Ig, human TROP2 Ig, human TREML2 Ig, mouse CTLA4 Ig fusion proteins - all expressed in NSO-cells – and recombinant human (rh) and recombinant mouse (rm) galectin-9 were purchased from R&D systems.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 1. Individual effects of BMP15 or GDF9 on preantral follicle growth in vitro. Follicles were mechanically isolated and cultured individually in 96-well plates for 72 h in a range of concentrations of BMP15 (a,b) or GDF9 (c,d) and with 10ng/ml FSH (b,d). Variation in follicle size and morphology exposed to different treatments are shown (e). Values are means SEM of 4-6 cultures. Comparisons between treatment groups were made within each time point by ANOVA and Bonferroni multiple comparisons test where *P .05 and **P .01 vs 0ng/ml.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: In Vitro, Isolation, Cell Culture

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 2. Combined effects of BMP15 and GDF9 on preantral follicle growth in vitro. Follicles were mechanically isolated and cultured individually in 96-well plates in BMP15 (250ng/ml), GDF9 (250ng/ml) or equal amounts (250ng/ml) of both and overall size relative to control was assessed at each time point (a). The co-operative effect of both ligands on follicle growth was assessed further using a fixed concentration of GDF9 (250ng/ml) and a range of concentrations of BMP15 (0-250ng/ml) (b). Values are means SEM (n 8 (a) and n 5 (b) cultures). Comparisons between treatment groups were made within each time point by ANOVA and Bonferroni multiple comparisons test where *P .05 and **P .01 vs control.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: In Vitro, Isolation, Cell Culture, Control, Concentration Assay

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 3. Effect of BMP15 and/or GDF9 on GC proliferation and apoptosis in cultured preantral follicles. Follicles were fixed after 24 or 48 h culture with BMP15 (250ng/ml), GDF9 (250ng/ml) or equal amounts (250ng/ml) of both. Sections of follicles were stained for Ki67 (green) or cleaved caspase-3 (red) and counterstained with DAPI (blue), examples at 24h are shown (a). Positive staining in the GC compartment was quantified for Ki67 and cleaved caspase-3 at 24h (b) and 48h (c). Values are means SEM (n 4-6 follicles/group). *P .05, **P .01 vs control (Cntrl). Scale bar represents 50 m.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: Cell Culture, Staining, Control

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 4. Candidate gene expression in cultured preantral follicles exposed to BMP15 and/or GDF9. Preantral follicles were cultured for 24 h in BMP15 (250ng/ml), GDF9 (250ng/ml) or equal amounts (250ng/ml) of both. Samples from each group consist of four pooled follicles that were snap frozen and analyzed by qRT-PCR. Relative gene expression was determined by normalization against Atp5b as described in Materials and Methods and expressed as fold changes relative to untreated control samples (Cntrl). Values are means SEM (n 4 ovaries/cultures). Groups with different letters are statistically different (P .05).

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: Gene Expression, Cell Culture, Quantitative RT-PCR, Control

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 6. Effect of dorsomorphin (DSM) on preantral follicle growth in the presence of BMP15. Follicles were cultured for 72 h in the presence of BMP15 (250ng/ml) or BMP15 (250ng/ml) DSM (1.0 M) and follicle size was assessed in relation to controls at each time point (a). In a separate experiment follicles were cultured for 48h in the presence of both BMP15 and GDF9 (250ng/ml each) and follicle size was assessed in relation to those cultured with both ligands and DSM (1.0 M) (b). Values are means SEM (n 4 cultures each). *P .05, **P .01 vs Control.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: Cell Culture, Control

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 7. Effect of SB505124 on preantral follicle growth in vitro. Follicles were mechanically isolated and cultured individually in 96-well plates for 72 h to test the effects of SB505124 on follicle growth in the presence or absence of GDF9 (a), BMP15 (b) or the combination of BMP15 GDF9 (c). All values shown are means SEM (n 6, 4 and 6 cultures for a, b and c respectively). Comparisons between treatment groups were made within each time point by ANOVA and Bonferroni multiple comparisons test. Groups with different letters are statistically different (P .05).

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: In Vitro, Isolation, Cell Culture