Figure 4.

Effects of hypoxia on low-density lipoprotein-induced unfolded protein response.

Notes: THP-1-derived macrophages were incubated for 72 hours with 200 μg/mL of low-density lipoproteins under hypoxia (1% oxygen) or normoxia (21% oxygen) or with thapsigargin at 200 nM for 20 hours under normoxia. (A) The abundance of phosphorylated eIF2α was assessed by Western blotting from total protein extracts with specific antibodies. β-actin was used as the loading control. The histogram shows the ratio of the intensity of the phosphorylated eIF2α band related to the intensity of the β-actin band as the mean ratio of three independent experiments ± standard deviation. *P<0.05 and ***P<0.001 versus normoxic control. (***)P<0.001 versus hypoxic control. [***]P<0.001 myeloperoxidase-modified versus copper sulfate-oxidized low-density lipoproteins. ^$$P<0.001 thapsigargin versus control using Student’s t-test. (B) For this XBP1 splicing assay, total RNA extracts were performed after 72 hours of incubation. Complementary DNA was then used as a matrix for a polymerase chain reaction using probes designed from both sides of the spliced intron. Polymerase chain reaction products were loaded on 1.5% agarose gel containing propidium iodide and pictures were taken after electrophoresis. The histogram shows the quantification of the bands on the agarose gel.

Abbreviations: CM(-LDL), cell-modified/native (low-density lipoproteins); CTL, control; H, hypoxia; Mox(-LDL), myeloperoxidase-modified (low-density lipoproteins); N, normoxia; Ox(-LDL), copper sulfate-oxidized (low-density lipoproteins); PeIF2α, phosphorylated eIF2α; Tha, thapsigargin.