Fig 1: DMT1 immunocytochemical staining in control tissues and B. malayi. A. Mouse CNS neuron staining positively for DMT1 (positive control) (bar = 5 µ). B. Negative control male B. malayi (without primary antibody) (bar = 70 µ). C. Uterine “sausage forms” are positive; the gut wall and the hypodermis are also positive (bar = 50 µ). D. Male worm showing spermatozoa (arrow) free of staining (Bar = 50µ). E. Strongly staining gut wall of a male worm (bar = 20 µ). F. Anterior end of a male worm: The wall of the pharynx and the hypodermis are moderately to strongly positive (bar = 50 µ).
Fig 2: The additional effect of transferrin (Holo) on miPS-LLCcm cells in vitro(A) Iron depletion and iron rich conditions were simulated by iron free medium and 1% or 15% FCS, respectively. Cell proliferation was measured using the XTT assay after incubation with transferrin for 48 hours at 37°C. Transferrin promoted the proliferation of miPS-LLCcm cells in iron depleted conditions (FCS1%), but not in iron rich conditions (FCS15%). Data are represented as average ± S.E.M. (n = 5). **: p < 0.01, ***: p < 0.001. (B) GFP subsets of miPS-LLCcm cells were measured by flow cytometry after incubation with transferrin for 48 hours at 37°C. Transferrin did not affect the GFP subsets of miPS-LLCcm cells. Data are represented as average ± S.E.M. (n = 5). (C) Normal and fluorescence microscopy findings showed that transferrin promoted the proliferation of GFP positive and negative cells. (D) Cultured miPS-LLCcm cells were treated with different concentrations of transferrin for 48 hours in iron depleted conditions. Cells were then harvested and total protein was analyzed for expression of the indicated proteins. Transferrin did not affect the expression of stemness markers and promoted the expression of TfR1 and DMT1. (E) Densitometry analysis of TfR1 and DMT1. The level of TfR1 and DMT1 expressed by non-treated cells was set at 100%. Data are represented as average ± S.E.M. (n = 3). *p < 0.05, **p < 0.01.
Fig 3: Western blot detection of divalent metal transporter 1 (DMT1) in Brugia malayi adult females. Soluble and membrane protein was extracted from B. malayi adult females (soluble protein lanes 1 and 6; membrane protein lanes 2 and 7) and HEK293 cells (soluble protein lanes 3 and 8; membrane protein lanes 4 and 9). In addition, a whole cell protein lysate was extracted from human Caco-2 cells (lanes 5 and 10). Approximately 20 µg of protein from each extract was subjected to SDS-PAGE followed by Western blot analysis with rabbit polyclonal anti-DMT1 antibody (Abcam, Cat: ab123085) (lanes 1 to 4) and rabbit polyclonal anti-GAPDH antibody (Proteintech, Cat: 10494-1-AP) (lanes 5 to 8).
Fig 4: Predicted transmembrane topology map of BmDMT1. The topology map was generated using the Protter server (Ottesen, 2000) and shows nine transmembrane regions and one N-glycosylation motif at amino acid position 348 (in green). The region corresponding to amino acids 261–291 of human DMT1 (NP_001167599.1) targeted by the anti-DMT1 antibody (Abcam, ab123085) is shown with identical, similar, and different residues highlighted. This region is 71% identical and 87% similar to human DMT1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 5: DMT1 immunocytochemical staining in O. volvulus. A. Microfilariae within egg shells are variably positive; the hypodermis (arrow) is also moderately positive (bar = 90 µ). B. Well-developed morulae are positive, while those under-developed or in transition are less positive. The gut wall (arrow) also is positive (bar = 80 µ). C. Stretched microfilariae are strongly positive (top right), early developing oocytes are lightly positive and the areas of the wall of the reproductive tract are strongly positive (bar = 70 µ). D. Emerged microfilariae in the outer areas of onchocercal nodules are DMT1 positive (arrow) (bar = 30 µ).
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