On performing Gene Set Enrichment Analysis (GSEA), it was found that platelet activation and growth as well as hemostasis were the most significantly enriched gene sets in low BMD condition, while Weighted Gene Coexpression Network Analysis (WGCNA) revealed that Annexin family genes including ANXA1, ANXA2, ANXA5
, and ANXA6 were upregulated in low BMD condition .
Liu et al., "Anxa5
mediates the in vitro malignant behaviours of murine hepatocarcinoma HcaF cells with high lymph node metastasis potential preferentially via ERK2/p-ERK2/c-Jun/p-c-Jun(Ser73) and E-cadherin," Biomedicine and Pharmacotherapy, vol.
Using a proteomics-based approach, which previously identified heme oxygenase-2 (HO-2) as [O.sub.2]-sensing protein partner of [BK.sub.Ca], we also identified ANXA5 as a potential interacting protein of [BK.sub.Ca] .
The aim of this study was to validate the interaction of ANXA5 and [BK.sub.Ca][alpha]-subunit ([BK.sub.Ca][alpha]), which was previously suggested by proteomics , to determine the biophysical consequences of the [BK.sub.Ca][alpha]/ANXA5 partnership, in particular to elucidate the structural elements required, and to define the impact of this partnership on apoptosis.
Knockdown and overexpression of ANXA5 were achieved by transient transfection of cells with specific siRNA and ANXA5 plasmid DNA, respectively.
[BK.sub.Ca][alpha] currents were recorded from inside-out membrane patches excised from the stably expressing the wild type HEK293 cells, transiently transfected with the ANXA5 siRNA or the ANXA5 plasmid.
HEK 293 [BK.sub.Ca] Cells and HEK 293 [BK.sub.Ca] cells transfected with ANXA5 siRNA (see above) were plated into 96-well white walled plates, in triplicate for each experimental condition.
Protein G Dynabeads (Invitrogen) was prepared following the manufacturers' instructions, 50 [micro]L of prepared Dynabeads was resuspended in 100 [micro]L citrate phosphate buffer (pH 5), and either 5 [micro]g of [BK.sub.Ca][alpha] (Alamone) or ANXA5 (Santa Cruz) antibody was added.
Considering all the genes in coagulation cascades (Figure 2(a)), the expression levels of 84.62% (11/13) of the genes were increased by more than 2-fold, with Anxa5
, Vwf, Serpine1, Ptgis, Thbd, Thbs1, Plg, and Plat being elevated by more than 6-fold (Figure 2(b)).
Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18).
The VPA-induced transcriptional induction of annexin A5 (Anxa5) and Anxa11 we detect in both embryos and cells (Table 2) may depend on HDAC inhibition, as the mouse Anxa5 and the human ANXA11 genes have Sp1 sites in their promoters (Bances et al.
By comparing gene expression changes in whole embryos with those in a cultured cell model, we defined a subset of VPA-responsive genes that may be evaluated as potential biomarkers of VPA teratogenicity (Lgals1, Idl, fasn, Anxa5).