Journal: eLife

Article Title: Hemozoin produced by mammals confers heme tolerance

doi: 10.7554/eLife.49503

Figure Lengend Snippet: ( A ) Structure of the SLC48A1 gene (which encodes SLC48A1) indicating the CRISPR target site in exon 1. ( B ) Predicted topology of SLC48A1 protein; arrow indicates the site of the two basepair deletion resulting in frameshift mutation. ( C ) Immunoblot analysis of membrane lysates prepared from spleens and livers of mice. Membranes were probed with anti-SLC48A1 antibody and then incubated with HRP-conjugated anti-rabbit secondary antibody. Each lane represents one animal. ( D ) SLC48A1 immunohistochemistry analysis of paraffin-embedded tissue sections of mice. Tissue sections were probed with affinity-purified anti-SLC48A1 antibody and then incubated with HRP-conjugated anti-rabbit secondary antibody. Images shown are representative of at least three mice. ( E–F ) Spleen wet weights and whole blood hematocrit from WT and KO mice. Each dot represents one mouse; mice were age (6 weeks) and sex-matched. ( G ) Representative images of spleens, livers and bone marrows of age and sex-matched mice. *p<0.05.

Article Snippet: RBCs were opsonized with the mouse red blood cell antibody (Rockland, cat. number 210–4139); 20 μl of antibody was used for approximately 10 9 RBCs.

Techniques: CRISPR, Mutagenesis, Western Blot, Membrane, Incubation, Immunohistochemistry, Affinity Purification

Journal: eLife

Article Title: Hemozoin produced by mammals confers heme tolerance

doi: 10.7554/eLife.49503

Figure Lengend Snippet: Quantifications of total Ter-119 + cells in the ( B ) bone marrow and ( E ) spleen. The %single cells* on the y-axis denote single cells that are negative for CD4/8/41, B220 and Gr-1. ( C ) Quantification of subpopulations of Ter-119 + cells represented as a percentage of total Ter-119 + cells in the bone marrow (n = 7–12). ( F ) Quantification of populations II and III of Ter-119 + cells represented as a percentage of total Ter-119 + cells in the spleen. Gating strategy ( G, I ) and quantification ( H, J ) of ( G, H ) splenic F4/80 hi Treml4 + red pulp macrophages (RPMs) and ( I, J ) F4/80 hi and F4/80 lo -CD11b hi splenic monocytes. At least 100,000 single cells were analyzed per sample. Each dot represents one mouse. **p<0.05; **p<0.01.

Article Snippet: RBCs were opsonized with the mouse red blood cell antibody (Rockland, cat. number 210–4139); 20 μl of antibody was used for approximately 10 9 RBCs.

Techniques:

Journal: eLife

Article Title: Hemozoin produced by mammals confers heme tolerance

doi: 10.7554/eLife.49503

Figure Lengend Snippet: ( A ) Experimental design of 59 Fe labeling and in vivo recycling. ( B ) Quantification of 59 Fe retained in tissues, represented as the ratio of the amount of radioactivity within an organ to that of the entire animal. ( C ) 59 Fe retained in differentially extracted fractions of the spleen at 96 hr, represented as counts per min. Total homogenate: homogenized and proteinase-treated whole spleen; Organic: ethyl acetate extractable [ 59 Fe]heme; Aqueous: ethyl acetate non-extractable 59 Fe; Insoluble fraction: proteinase-insoluble fraction containing 59 Fe (n = 4–6 across all groups and timepoints). ( D ) Image of insoluble fractions before and after dissolving in buffer containing 100 mM NaOH, 2% SDS and 3 mM EDTA. ( E ) Visible spectra of dissolved insoluble fractions. ( F ) Powder x-ray diffraction of purified insoluble fraction from KO spleens (red: measured data; dark blue: calculated pattern; light blue: background; green: structural plot) ( G ) Chemical structure of hemozoin from KO mice. ( H ) Quantification of splenic heme by spectrophotometric measurements (n = 3). ( I ) Scanning electron microscopy of hemozoin isolated from Plasmodium falciparum , KO spleen, liver, and bone marrow. ( J ) Transmission electron microscopy of F4/80 + bone marrow and splenic macrophages from WT and KO mice. At least 3 cells were imaged per genotype. ( K ) Confocal microscopy of bone marrow macrophages from WT and KO mice probed with anti-LAMP1 antibody and secondary alexa-488 antibody. Hemozoin is pseudocolored as orange. White arrow points to hemozoin-laden vesicle, yellow arrow points to non hemozoin-laden vesicle. At least 20 cells were analyzed per genotype. *p<0.05; **p<0.01.

Article Snippet: RBCs were opsonized with the mouse red blood cell antibody (Rockland, cat. number 210–4139); 20 μl of antibody was used for approximately 10 9 RBCs.

Techniques: Labeling, In Vivo, Radioactivity, Purification, Electron Microscopy, Isolation, Transmission Assay, Confocal Microscopy