and N.Z.; supervision, N.Z.; funding acquisition, N.Z. over 95% of the Mn uptake from the basolateral side [21]. In kidney proximal tubule cells, the knockdown of either ZIP14 or ZIP8 was associated with a significant decrease in Mn uptake [28]. In mouse fibroblasts (MEF), ZIP8 overexpression resulted in elevated cellular Mn accumulation; moreover, in MEF cells, ZIP8 had Ctsd a higher affinity for Mn than BSI-201 (Iniparib) all other trace metals tested, except for cadmium [29]. In HEK293 cells, ZIP8 was detected on the plasma membrane and ZIP8 overexpression increased cellular Mn accumulation [30]. Together, these studies clearly demonstrate that both ZIP14 and ZIP8 are Mn importers. Similarly to the epithelial cells of the intestine and liver, HIBCPP cells form a polarized monolayer with specific apical and basolateral orientation [5,31,32]. Identifying Mn transporters in these cells will help us to understand the mechanisms of Mn homeostasis within the brain. Whether ZIP14 and ZIP8 are expressed in HIBCPP cells and whether these two Mn transporters are localized to apical or basolateral plasma membranes are not known. In the present study, we identified that both ZIP14 and ZIP8 are expressed in HIBCPP cells and that both transporters are involved in cellular Mn uptake. In addition, we found that ZIP14 and ZIP8 are enriched at opposite sides of HIBCPP cells. These results provide novel insights for the understanding of potential Mn transport mechanisms in HIBCPP cells. 2. Materials and Methods 2.1. Cell Culture HIBCPP cells were maintained in DMEM/F12 (Corning Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum, 100 g/mL streptomycin, 100 units/mL penicillin, 4 mM L-glutamine, and 5 g/mL insulin. To be consistent with previously published results using HIBCPP cells, only cells before passage 37 were used for experiments [33]. HEK293 and A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 g/mL streptomycin, and 100 units/mL penicillin. All cells were grown in a humidified incubator at 37 C with 5% CO2. Media was changed every 2C3 days and trypsin (0.05% in phosphate buffered saline [PBS]) (Life Technologies, Grand Island, NY, USA) or Accutase (Corning Inc., Corning, NY, USA) was used to detach cells for passaging. 2.2. RNA Isolation, PCR, Quantitative Real-Time PCR (qRT-PCR), and Melting Curve Analysis Total RNA was isolated from HIBCPP cells using the Quick-RNA MiniPrep Plus kit (Zymo, Irvine, CA, USA). Complementary DNA was synthesized from isolated RNA using M-MuLV Reverse Transcriptase (New England BioLabs, Ipswich, MA, USA). To verify the primers, PCR was performed using a Direct PCR kit (Bimake, Houston, TX, USA) and a thermal cycler (C1000 Touch Thermal Cycler, Bio-Rad, Hercules, CA, USA). The primers used were as follows: for 10 min. The supernatant containing the whole lysate was transferred to 1.5 mL collection tubes. An aliquot of 300 L was stored at ?80 C for future analysis, while the remaining lysate was transferred to a Pierce centrifuge column pre-loaded with 100 L of Pierce high capacity NeutrAvidin agarose beads (ThermoFisher Scientific, Waltham, MA, USA), per the manufacturers protocol. Samples were incubated for 2 h at 4 BSI-201 (Iniparib) C on a rocker. The column was centrifuged for 1 minute at 1500 to collect the flow-through. The beads were washed three times with TBS containing protease inhibitors. After each wash, the column was centrifuged for 1 minute to remove the flow through. To elute the biotinylated proteins, 100 L of 1 1 sample buffer (1.7% (to collect the surface protein fraction. This membrane protein fraction was stored at ?80 C for further Western blot analysis. 2.7. Manganese Uptake 54Mn uptake was measured in HIBCPP cells grown on 6-well plates for 72 h. The uptake procedure was performed as previously described [21]. Briefly, two-times concentrated (0.2 M) 54Mn solutions were prepared from 54MnCl2 (PerkinElmer Inc., Waltham, MA, USA) complexed to citrate on the day of the experiment. Higher BSI-201 (Iniparib) concentrations of Mn were achieved by adding Mn citrate in the required amounts. During the experiments, cells were washed three times with PBS+/+ and pre-incubated in 1 mL of transport medium (DMEM with 1 mM pyruvate and 20 mM HEPES, pH 7.4) at 37 C (or 4 C) in a humidified BSI-201 (Iniparib) incubator (or on ice) for 30 min. 54Mn uptake experiments were initiated by adding 1 mL of.