Inflammation and ER Stress Downregulate BDH2 Expression and Dysregulate Intracellular Iron in Macrophages

Figure 1

ER stress and inflammation downregulate BDH2 expression in macrophages. (a) Western blot analysis of BDH2 protein expression in THP-1 macrophage-like cells ( cells/mL) treated with tunicamycin (10 μg/mL) or vehicle (DMSO) overnight prior to LPS exposure (40 ng/mL) for 6 hr. BDH2 protein was detected with monoclonal anti-BDH2 TrueMAB antibody clone 2G. Lane 1: vehicle-treated THP-1 cells; 2: tunicamycin treated; 3: LPS treated; 4: tunicamycin and LPS treated; 5: untransfected HEK293 cells as negative control; 6: BDH2 transfected HEK293 cells as positive control (BDH2 positive control contains a FLAG tag and 10-fold higher concentration was loaded in panel (i) compared to panel (ii), representing two independent experiments); band. (b) THP-1 macrophages were treated with tunicamycin (10 μg/mL) as above and RNA was extracted and BDH2 gene expression was assessed by quantitative RT-PCR normalized to that of β-actin. (c) THP-1 cells treated with tunicamycin doses 1, 2.5, or 5 μg/mL and incubated overnight. The fold change in BDH2 gene expression was calculated in reference to DMSO treated control THP-1 cells using the ΔΔCT method and presented here normalized to vehicle (DMSO) treatment control. Error bars represent the SD from the mean of nine readouts generated from three independent experiments. values were calculated in reference to vehicle (DMSO) treatment control using one-way ANOVA followed by Bonferroni multiple comparisons post hoc analysis. values annotated ; ; were not significant (NS).