Fig. 4.

Bafilomycin A1 prevents Mn directed ZIP14 degradation. HepaRG cells were pretreated with 50 nM bafilomycin A1 (Baf A1) for 30 minutes prior to and during incubation with 50 μM Mn for 4 additional hours. Top panel: Western analysis indicates ZIP14 levels are similar to control with Baf A1 treatment. Shown is a representative blot. Equal volumes of lysed samples were loaded per lane. ZIP14 band intensity was measured and normalized to GAPDH levels. Values are means ± SEM, n=4; a, significant from control. P < 0.05. Bottom panel: Shown are representative staining examples for ZIP14 (green) and Lamp1 (red) in the same treatment groups using indirect immunofluorescence. Below each image is an insert from the region indicated (white inset). IF experiments were performed in triplicate on two separate occasions. Image analysis was performed using ImageJ software and the JaCoP plugin to determine the Pearson’s coefficients expressed as means ± SD: Control = 0.275 ± 0.020, Mn = 0.234 ± 0.018, and BafA1+Mn = 0.364 ± 0.039. Pearson values for Bafilomycin A1-treated cells were significantly different from HepaRG cells in control conditions (P = 0.0004) and Mn-treated cells (P < 0.0001). Mn-treated cells were significantly different from control (P = 0.0045).