McDermott JR , Geng X , Jiang L , Gálvez-Peralta M , Chen F , Nebert DW , Liu Z .

R15 ES022800 NIEHS NIH HHS , P30 ES006096 NIEHS NIH HHS , R01 ES020137 NIEHS NIH HHS , R01 ES010416 NIEHS NIH HHS , T32 ES016646 NIEHS NIH HHS

	Figure 1. Intracellular Se content and toxicity; ZIP8 protein levels in human prostate cancer cellsA. effect of Zn2+ on Se content in selenite-treated DU145 cells; Zn2+ added concomitantly with HSeO3− for 30 min. B. effect of Zn2+ on selenite-mediated viability of DU145 cells following 12-h treatment, varying HSeO3− amounts. Untreated cultures were given a ratio value of 1.0., and other ratios of experimental regimens are expressed, relative to 1.0. C. effect of EDTA and Zn2+ on Se content in selenite-treated cells; EDTA (5 mM) + Zn2+ (200 μM) pretreatment was 5 min; at far right, Zn2+ was washed out prior to HSeO3− treatment for 30 min. D. Western blots of human ZIP8 in five prostate cancer lines (left lanes) and five prostate non-cancer cell lines (right lanes). GAPDH, glyceraldehyde 3-phosphate dehydrogenase, lane-loading control. E. cell viability of Zn2+-treated ZIP8-MEFs versus LUC-MEFs, as a function of selenite concentration; treatment (5 μM Zn2+ and 25 mM HCO3−) was 16 h. For panels B and E, “relative viability” of cells not treated with selenite is given a value of 1.0, and viability of all other experimental regimens are expressed relative to that control.
	Figure 2. Se uptake as a function of time and Zn2+ concentration in oocytes and mouse cell cultures; localization of ZIP8 to plasma membraneA. Se content following addition of Zn2+ (500 μM), or Zn2+ + HCO3− (3.5 mM) in selenite-treated Xenopus laevis oocytes expressing mouse ZIP8 cDNA (closed bar) versus control oocytes carrying vector only (open bar). Without added HCO3−, note that a basal level of HCO3− exists in the ND96 medium due to pCO2 in solution, as can be calculated by the Henderson-Hasselbach equation [30]. B. Se uptake kinetics in stably-transfected high-ZIP8-expressing ZIP8-MEFs vs control low-ZIP8-expressing LUC-MEFs during 20-min exposure to 50 μM HSeO3− + 100 μM Zn2+. C. Intracellular Se content in ZIP8-MEFs as a function of HSeO3− concentration; cells were treated with Zn2+ (100 μM) plus 5 to 100 μM HSeO3−. D. Intracellular Se content in ZIP8-MEFs (treated with 50 μM HSeO3− for 20 min) as a function of Zn2+ concentration. E. Intracellular Se content as a function of HSeO3− concentration (20-min treatment) in wild-type (WT) vs Slc39a8(neo/neo) knockdown mouse primary embryonic fibroblast (MEF) cultures; Zn2+ (100 μM) and HCO3− (25 mM) were constant. F. confocal microscopy of co-localization of ZIP8 (green) with the membrane marker Na+/K+-ATPase subunit (red) in ZIP8-MEF versus LUC-MEF cultures. DAPI, blue stain for DNA, i.e. nucleus. For all mouse culture experiments, HCO3− was present in culture medium at 25 mM. Addition of the HCO3− concentrations used in these experiments in either frog oocytes or mouse cultures did not significantly alter pH (7.5) of the medium. Brackets denote S.D. *P <0.05, **P <0.01.
	Figure 3. ZIP8 knockdown by shRNA causes decreased ZIP8 protein expression and Se and Zn uptakeA. Western blot of human ZIP8 in TAs cells stably expressing shRNA against ZIP8 mRNA (shRNA-1 and shRNA-2) vs scrambled shRNA (sc-shRNA) control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase, lane-loading control. B. Se content and C. Zn content in TAs cells, ZIP8-knockdown vs control, after exposure to HSeO3− + Zn2+ (both 200 μM) for 30 min. TAs_ZIP8-shRNA-1 and TAs_ZIP8-shRNA-2 represent two different stably-expressing cell lines carrying two different shRNAs. For all experiments, HCO3− (25 mM) was present in the culture medium. Intracellular Se and Zn content was determined by ICP-MS. Brackets denote S.E.M. *P <0.05, **P <0.01.
	Figure 4. Se content in ten mouse tissues as a function of ZIP8 concentrations in three mouse lines having different Slc39a8 genotypesA. Intracellular Se content, 2 h after IP selenite (2.5 mg/kg) administration to the ZIP8-over-expressing BTZIP8-3 mouse line (solid red bar; N=6), the ZIP8-normal-expressing wild-type (WT; pink bar; N=9), and the hypomorph ZIP8 Slc39a8(+/neo) line; (open bar; N=9). Red brackets (S.E.M.) depict statistical differences between BTZIP8-3 and Slc39a8(+/neo); black brackets (S.E.M.) signify statistical differences between BTZIP8-3 and WT. *P <0.05, **P <0.01. Animals (2-3 months of age) included similar numbers of males and females; we first had determined that no sex differences existed. B–F. ZIP8 immunofluorescence in 10-μm tissue sections from WT vs BTZIP8-3 mice. White bar (in panel B) = 50 μm for panels B through E; white bar (in panel F) = 10 μm.
	Figure 5. Se content in mouse whole blood or washed red blood cells (RBCs)A. relative Se content in WT whole blood, following HSeO3− treatment (20 μM; 15 min at 37°C), with, versus without, Zn2+ (75 μM); where indicated, whole blood was pretreated for 5 min with EDTA (4 mM) or TPEN (100 μM). Value for selenite-treated whole blood without added Zn2+ was chosen as “1.0” and all other values are relative to that. B. Intracellular Se content in isolated WT RBCs resuspended in PBS containing HCO3− (10 mM; 15 min at 37°C), as a function of Zn2+ concentration following 5-min pretreatment with HSeO3− (20 μM, 37°C). C. Intracellular Se content in isolated RBCs from the three mouse genotypes resuspended in PBS with HCO3− (25 mM) + 75 μM Zn2+ (closed red bars) versus 25 mM HCO3− alone (open bars). Cells were incubated with HSeO3− (20 μM) for 5 min at 37°C. **P <0.01, comparing BTZIP8-3 with, vs without, added Zn2+. D. comparison of rate of Se uptake in RBCs (also resuspended in PBS), as a function of incubation time (37°C); RBCs were isolated from BTZIP8-3, WT and Slc39a8(+/neo) mice; RBCs were treated with HSeO3− (20 μM). **P <0.01, comparing BTZIP8-3 with both WT and Slc39a8(+/neo). Brackets denote S.E.M.
	Figure 6. Intracellular Se content of RBCs (resuspended in PBS, without any exogenous Zn2+ or HCO3−), in response to thiol, HSeO3−, and/or EDTARBCs were incubated for 2 h with HSeO3− or its reduced products (20 or 100 μM): first two bars denote first being reduced for 15 min by GSH (100 μM) alone, causing build-up of extracellular reduced selenium product(s) (derived from 20 μM HSeO3−, or with GSH plus EDTA (5 mM); second two bars indicate no reduction by GSH first, but higher concentrations of HSeO3− treatment (100 μM), comparing GSH (800 μM) with or without 5-min EDTA pretreatment (5 mM); last two bars designate HSeO3− treatment (20 μM) alone or with 5-min EDTA (5 mM) pretreatment, but without GSH. *P < 0.05, **P < 0.01.
	Figure 7. The two proposed models of selenite transportA. ZIP8-mediated HSeO3− uptake model, which is both zinc- and bicarbonate-dependent. Right, the extracellular HSeO3− conversion model. In the former, ZIP8-mediated HSeO3− uptake can be prevented with prior chelation of Zn2+ by EDTA. Intracellular HSeO3− reacts spontaneously with proteins containing coordinated thiol groups−−modifying signaling pathways dependent on zinc-finger proteins, or thiol proteins. Functional downstream effects on multiple targets are dependent on HSeO3− concentration and ZIP8 levels. HSeO3− reactions with zinc-finger proteins might release free Zn2+, which, in addition to Zn2+ transported by ZIP8, is proposed to elevate labile Zn2+ concentrations and participate in subsequent zinc-signaling functions. Concentration-dependent effects (bottom) range from anti-cancer and anti-inflammatory, to apoptosis and oxidative stress, to cytotoxicity. B. In the alternative model, HSeO3− uptake is proposed to be regulated by extracellular thiol reduction of selenite–in the extracellular milieu–followed by uptake of unknown reduced Se product(s) [22, 35].

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