Journal: Cells

Article Title: FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells

doi: 10.3390/cells9091927

Figure Lengend Snippet: Pancreatic differentiation in presence of growth factors. ( a ) Gene expression of PDX1 , HLXB9 , HNF1B , HNF6 , SHH, TBX1, SOX9, AFP, and CDX2 measured by RT-qPCR in human embryonic stem cells (hESC)s at d0, d4, and d8 of differentiation treated ± 5 or 50 ng/mL fibroblast growth factor (FGF)2, FGF7, FGF10, or epidermal growth factor (EGF) in stage 2 medium. Values are means ± SEM, n = 4–6. 5/50 = 5 or 50 ng/mL growth factor, C = control medium without growth factors. ( b ) Fluorescence micrographs after pancreatic differentiation illustrating the protein expression of PDX1/HNF1B after a 4-day treatment ± 5 or 50 ng/mL FGF2, FGF7, FGF10, and EGF. Nuclei were counterstained with DAPI. Scale bar = 200 µm and 20 µm for higher magnification of the control. ( c ) Quantification of cell expansion using ± 50 ng/mL growth factor. Data are presented as mean (log2) ± SEM, n = 3. ( d ) Quantification of PDX1-positive cells upon treatment ± 5 or 50 ng/mL FGF2, FGF7, FGF10, and EGF at d8. Percentages are expressed as means ± SEM. *** = p ≤ 0.001, ** = p ≤ 0.01 compared to control, ANOVA plus Dunnett’s post-test.

Article Snippet: The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used.

Techniques: Gene Expression, Quantitative RT-PCR, Control, Fluorescence, Expressing

Journal: Cells

Article Title: FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells

doi: 10.3390/cells9091927

Figure Lengend Snippet: Gene expression changes in response to different FGF2 concentrations. ( a ) hESCs were cultured in the presence of 5–50 ng/mL FGF2 during differentiation into PDX1-positive cells and compared to a control condition (=C). Gene expression changes of PDX1 , HNF1B , SHH, and TBX1 are presented as means ± SEM, n = 5–12. *** = p ≤ 0.001, ** = p ≤ 0.01, * = p ≤ 0.05 compared to control, ANOVA plus Dunnett’s post-test. ( b ) Effect of SHH inhibition by 10 µM Gant 58 or 2.5 µM Sant1 in stage 2 medium ± 50 ng/mL FGF2. Gene expression changes of PDX1 and SHH are presented as means ± SEM, n = 4–5. *** = p ≤ 0.001, compared to control, ANOVA plus Dunnett’s post-test.

Article Snippet: The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used.

Techniques: Gene Expression, Cell Culture, Control, Inhibition

Journal: Cells

Article Title: FGF2 Inhibits Early Pancreatic Lineage Specification during Differentiation of Human Embryonic Stem Cells

doi: 10.3390/cells9091927

Figure Lengend Snippet: Inhibition of FGFR1c/3c rescues pancreatic development in presence of FGF2 and FGF7. ( a ) Gene expression changes of PDX1 , HNF1B , SHH , and TBX1 in d8 cells treated ± 10 ng/mL FGF2 (F2) and the FGFR1c/3c small molecule inhibitor PD-173074 (PD) with a concentration of 100 nM (PD). Data are means ± SEM, n = 4–5. *** = p ≤ 0.001, ** = p ≤ 0.01, * p = ≤0.05 ANOVA plus Tukey’s post-test, F2 treatment compared to F2 + PD and PD only. ( b ) Representative immunofluorescence staining of PDX1 in d8 control cells, cells treated with F2 and F2 + PD. Nuclei were counterstained with DAPI. Scale bar = 100 µm. ( c ) Gene expression changes of PDX1 , SOX9 , AFP , and CDX2 in d8 cells treated ± 50 ng/mL FGF7 (F7) and ± PD-173074 with a concentration of 10 or 100 nM (10, 100). Values are means ± SEM, n = 3–6, * = p ≤ 0.05, ANOVA plus Tukey’s post-test. ( d ) Quantification of PDX1-positive cells at d8 upon treatment ± 50 ng/mL FGF7 and ± PD-173074 with a concentration of 10 or 100 nM. Percentages are expressed as means ± SEM. * = p ≤ 0.05, ANOVA plus Tukey’s post-test.

Article Snippet: The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used.

Techniques: Inhibition, Gene Expression, Concentration Assay, Immunofluorescence, Staining, Control

Journal: PLoS ONE

Article Title: Inhibition of Uterine Sarcoma Cell Growth through Suppression of Endogenous Tyrosine Kinase B Signaling

doi: 10.1371/journal.pone.0041049

Figure Lengend Snippet: (A) Messenger RNA expression of TrkB, p75NTR and their ligands was detected by RT-PCR in the uterine sarcoma cell lines, MES-SA and MES-SA/Dx5. As loading controls, the levels of β-actin were also assessed. The negative control lacked template DNA. (B) Messenger RNA expression of TrkB, p75NTR, and their ligands, BDNF and NT4/5, in surgical specimens from human uterine leiomyosarcomas was detected by RT-PCR. As loading controls, β-actin mRNA levels were assessed. (C) Expression levels of TrkB, BDNF and NT4/5 in uterine sarcoma cell lines were quantified by real-time RT-PCR. The expression level of each transcript was standardized using levels of β-actin transcripts in the same samples (n = 4). Columns , mean; bars , SE. *, P <0.05 vs. MES-SA. (D) Immunohistochemical detection of TrkB, NT4/5 and BDNF proteins. Target proteins (red signal) were detected in MES-SA/Dx5 cells (Upper panels). Lower panels depict negative controls. Nuclei (blue) were stained with Hoechest 33342. (Scale bars, 50 µm).

Article Snippet: The human uterine sarcoma cell lines, MES-SA and MES-SA/Dx5, and human uterine leiomyosarcoma cell line, SKN were purchased from the American Type Culture Collection (Manassas, VA, USA) and the Japan Health Science Foundation (Osaka, Japan), respectively.

Techniques: RNA Expression, Reverse Transcription Polymerase Chain Reaction, Negative Control, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Inhibition of Uterine Sarcoma Cell Growth through Suppression of Endogenous Tyrosine Kinase B Signaling

doi: 10.1371/journal.pone.0041049

Figure Lengend Snippet: To determine the effects of endogenous TrkB ligands on cell proliferation (A) and survival (B), uterine sarcoma cells (MES-SA/Dx5 cells) were cultured in medium alone (control, C), or with different doses of the TrkB ectodomain (TrkB EC), the pan-Trk inhibitor, K252a, or its inactive analogue, K252b. Cell proliferation activity was determined using the cell proliferation reagent WST1 after 48 h of culture, and cellular apoptosis was quantified using the caspase-3/7 assay after 8 h of culture (n = 3). Columns , mean; bars , SE. *, P <0.05 vs. control. (C) DNA fragmentation was detected by in situ TUNEL staining. Nucleic acids are stained with propidium iodide (red). Representative images were obtained from MES-SA (upper panels) and MES-SA/Dx5 (lower panels) cells after treatment ± K252a (1 µM) or K252b (1 µM). The number of apoptotic cells (green) was increased in the cells with K252a treatment. (Scale bars, 100 µm).

Article Snippet: The human uterine sarcoma cell lines, MES-SA and MES-SA/Dx5, and human uterine leiomyosarcoma cell line, SKN were purchased from the American Type Culture Collection (Manassas, VA, USA) and the Japan Health Science Foundation (Osaka, Japan), respectively.

Techniques: Cell Culture, Control, Activity Assay, In Situ, TUNEL Assay, Staining