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