Supplementary Materialsnutrients-11-01473-s001. Mn uptake by A549 cells. To our surprise, knockdown of either ZIP8 or ZIP14 impaired Mn accumulation to a similar extent, which we traced back to comparable amounts of ZIP8 and ZIP14 at the plasma membrane. Our study highlights the importance of both ZIP8 and ZIP14 in Mn metabolism of alveolar epithelial cells. 0.05, ** 0.01, and *** 0.001. 0.05 was considered as not significant. The PRISM 5 software (GraphPad, La Jolla, CA, USA) was used for statistical analysis. 3. Results 3.1. A549 Cells Express Higher Levels of ZIP8 than ZIP14 The alveolar epithelium consists of Type I and Type II cells [17]. As these cells may differ in their transporter expression, we first assessed whether A549 cells, a Type II alveolar cell collection, provided a suitable model to study the function of ZIP8 and ZIP14 in the alveolar epithelia. In humans, ZIP8 is usually most abundantly expressed in the lungs and at a much lower levels in the intestine, kidney, and liver, whereas ZIP14 expression levels are highest in the intestine and liver but very low within Phenformin hydrochloride the lungs [25,35]. We likened the proteins and mRNA degrees of ZIP8 and ZIP14 in A549 Phenformin hydrochloride cells with those in CaCo-2, HEK293, and HepG2 cells that people utilized as cell versions for the intestine, kidney, and liver organ, respectively. Real-time PCR and immunoblot analyses uncovered that A549 cells portrayed the highest levels of ZIP8 one of the cell lines examined, at both mRNA and proteins Rabbit Polyclonal to PITX1 levels (Body 1ACC and Body S2A,B). On the other hand, A549 cells included only low degrees of ZIP14 mRNA and proteins in comparison to CaCo-2 and HepG2 cells (Body 1DCF and Body S2C,D). Open up in another window Body 1 Appearance of ZIP8 and ZIP14 in individual cell lines. (A) ZIP8 and (D) ZIP14 Phenformin hydrochloride mRNA duplicate amount of A549, CaCo-2, HEK293, and HepG2 cells. Data are provided as means SD from three indie experiments. Statistical evaluation was performed using one-way ANOVA accompanied by the Bonferroni post-hoc check with * 0.05, ** 0.01, and *** 0.001. (B) ZIP8 and (E) ZIP14 immunoblot of whole-cell lysates of A549, Phenformin hydrochloride CaCo-2, HEK293, and HepG2 cells. Both GAPDH and -ACTIN were used as launching controls. Specific rings for ZIP8 and ZIP14 (monomers and multimers) are indicated by arrows. Particular levels of (C) ZIP8 and (F) ZIP14 (monomeric + multimeric forms) in A549, CaCo-2, HEK293, and HepG2 had been determined utilizing the fusion protein that were useful for the era from the ZIP8- and ZIP14-antibodies as quantitative markers (find Body S3 for information). Data are provided as means SD from four indie experiments. Statistical evaluation was performed using one-way ANOVA accompanied by the Bonferroni post-hoc check with * 0.05, ** 0.01, and *** 0.001. The appearance profile of both ZIP14 and ZIP8 in A549, CaCo-2, HEK293, and HepG2 cells shows the organ-specific appearance in vivo. Hence, A549 cells could be seen as a valid model to review the features of ZIP8 and ZIP14 within the alveolar epithelia. In A549 cells, the duplicate number of ZIP8 mRNA were about 23 Phenformin hydrochloride occasions that of ZIP14 mRNA (Number 1A,D), suggesting that ZIP8 was strongly enriched over ZIP14 in these cells. To allow for a direct assessment of ZIP8 and ZIP14 protein levels, we identified the specific amounts of both transporters utilizing the fusion-peptides used to generate the respective antibodies as quantitative markers (Number S3). Consistent with the outcome from the.