Fig 1: The effect of TNF and ATA exposure on HUVEC and fibroblast expression levels of THBD, PROCR, and TFPI, and HUVEC expression of F3, F9, and F10. The changes in gene expression of the surface coagulation regulatory proteins were measured in untreated HUVECs and fibroblasts (black), cells exposed to 10 ng/ml TNF (red), and cells exposed to 30 µg/ml ATA (blue) for 24 hours. Graphs show fold-changes in expression of (a) THBD, PROCR, and TFPI and (b) F3, F9, and F10 in HUVECs and (c) THBD, PROCR, and TFPI in fibroblasts (means + SD). Number of gene measurements per experimental condition, HUVECs: THBD, n = 6; PROCR, n = 4–5; TFPI, n = 3; F3, n = 3–4; F9, n = 4; F10, n = 3; for fibroblasts, each gene was measured 4 times under each experimental condition. *p < 0.05.
Fig 2: Regulation of FX protein expression. (A) GBM and normal brain tissue (NBT) were sectioned and probed for FX in neurons identified by NeuN and glial cells identified by GFAP using specific antibodies (bar = 50 μm) and (B) the overall expression of FX in these tissues was quantified by fluorescence intensity using an image scan (n = 3, t-test). (C) U87 cells were cultured in the condition of hypoxia and serum starvation (i.e., OGD: oxygen and glucose depletion) for different times at 37 °C. They were then processed for quantifying F10 mRNA (n = 3, one-way ANOVA). (D) Cultured U87 cells were stimulated with increasing doses of LPS for 24 h and then processed for immunoblot with an FX antibody and quantified using densitometry (left panel: a representative immunoblot image, right panel: a summary of 3 independent experiments (one-way ANOVA). (E) RT-qPCR was performed to quantify levels of the F10 mRNA in U87 cells after stimulation with LPS (n = 3, one-way ANOVA). For all figures, * p < 0.05, ** p < 0.01, **** p < 0.0001.
Fig 3: F10 transcript analysis. (A) Coding exons included in the different isoform mRNAs of F10. Blue boxes represent exons. Due to the missing nucleotide sequence in exon 7 (yellow), translation was terminated prematurely when it reached exon 8 (red arrow moved forward). (B) Variants identified by Snapgene: F10 mRNA transcripts were cloned from healthy human brain tissue and glioblastoma tissue using TA cloning. The base deletion in exon 7 of the mRNA transcript 3 is shown in the lower panel, and the upper panel shows bases at the same position in transcript 1. (C) The ratios of the two variants in 4 normal healthy brain samples and 7 glioblastoma samples. * p < 0.05.
Fig 4: Expression of FX in glioma tissues and cell lines. (A) F10 mRNA levels in cells from the GBM cell lines U87, U251, and SNB-19 relative to the levels in HepG2 cell (n = 3, one-way ANOVA). HepG2 cells from the liver were used as a positive control. (B) RT-qPCR showed that F10 mRNA levels decreased in F10-knockdown U87 cells (sh-F10-1, sh-F10-2, sh-F10-3, n = 3 for each shRNA construct). (C) Detection of F10 transcripts in GBM specimens, healthy brain tissue (NBT: normal brain tissue), and human liver tissue (HLT: human liver tissue). Representative immunoblots show (D) FX protein expressed in cells from the three clonal glioblastoma lines of U251, SNB19, and U87 and (E) FX in glioblastoma tissue samples. HepG2 cells were tested as positive controls. (F) The synthesis of FX protein was suppressed in glioblastoma U87 cells sh-FX-1, sh-FX-2, and sh-FX-3 (n = 3 for each shRNA construct). The quantitative data were analyzed using one-way ANOVA, *** p < 0.001, ** p < 0.01.
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