Figure 5.

Expression of SLC30A10-WT or SLC30A10-T95I in ΔSLC30A10 HepG2 cells rescues changes in Mn levels and cell viability. A: amino acid sequence of the wild-type (WT) SLC30A10 protein or the SLC30A10 protein sequence translated from the genomic DNA sequence of the ΔSLC30A10 HepG2 clone. There are two sequences (Seq1 and 2) for the ΔSLC30A10 clone because CRISPR/Cas9 introduced two independent mutations in the corresponding genomic DNA, likely one for each chromosome. Differences between the sequences obtained from the ΔSLC30A10 clone and the WT protein are shown in red. Numbers indicate amino acid residue. B: RT-PCR analyses were performed to detect SLC30A10 or TBP mRNA in control or ΔSLC30A10 HepG2 cells. C–F: intracellular metal measurements were performed after 125 µM Mn treatment for 16 h in mock-infected HepG2 cells (control) or ΔSLC30A10 HepG2 cells that underwent a second mock, SLC30A10-WT or SLC30A10-T95I infection (ΔSLC30A10 + control; ΔSLC30A10+SLC30A10-WT; or ΔSLC30A10+SLC30A10-T95I, respectively). Clonal selection was not performed after SLC30A10-WT or SLC30A10-T95I infection. Metal values were normalized to total cell counts (means ± SE; n = 8, *P < 0.05 and n.s. denotes not significant for indicated comparisons by one-way ANOVA and Tukey’s post hoc test). G: viability of cells infected as described in C–F was assayed after 16 h of treatment with indicated Mn doses. For each infection condition, viability at 0 mM Mn was set to 100 (means ± SE; n = 5, *P < 0.05 using two-way ANOVA and Tukey’s post hoc test with a, b, and c indicating differences in comparison with control, ΔSLC30A10 + control, or ΔSLC30A10+SLC30A10-T95I infection groups, respectively at each Mn concentration). H: RT-PCR analyses were performed in cells infected as described in C–F to detect SLC30A10 or TBP mRNA. Cont., control.