Hagfish VWF forms multimers in mammalian cell culture. (A) qPCR analysis of hagfish vwf in untransfected CHO cells (CHO WT) and CHO cells stably transfected with hagfish vwf (clones 18 and 23). mRNA expression is represented as copy number per 10⁶ 18S copies. Data are presented as mean ± SD (n = 3 fish). (B) Heterologous expression of hagfish Vwf in CHO cell culture visualized by western blotting of whole-cell lysates on reducing or nonreducing SDS-PAGE with B10 anti-hagfish mAb or Dako anti-human pAb, as indicated. (C) Representative confocal laser scanning microscopy images of control CHO cells (CHO WT) or CHO cells stably transfected with hagfish Vwf (CHO18 Hagfish Vwf) processed for staining of Vwf using polyclonal anti-human VWF antibody (Dako) or monoclonal anti-hagfish Vwf antibody (B10). All stain and magnification information for photomicrographs is provided in the supplemental Material. (D) Electron micrographs of control CHO cells (CHO WT) and CHO cells expressing hagfish tissue factor pathway inhibitor (CHO Hagfish Tfpi) or hagfish Vwf (CHO18 Hagfish Vwf). Nucleus appears at the top of each cell. Note the large irregularly shaped organelles (arrowheads) in the Vwf-expressing cells which contain electron dense material.