Figure 9.

JNK2 is necessary for apoptosis, but activation is not dependent on SRA engagement. (A) Macrophages were preincubated for 30 min with 10 μM SP600125 or with vehicle and then incubated for 18 h in medium alone or medium containing ac-LDL plus 58035 ± SP600125. The cells were stained with Alexa 488 Annexin V (green) and propidium iodide (red). Representative fluorescent images and quantitative apoptosis data from four fields of cells for each condition are shown. The data are expressed as the percent of total cells that stained with Annexin V and propidium iodide. Data are expressed as mean ± SEM (n = 4). Bar, 25 μm. (B) Macrophages were preincubated for 30 min with 15 μM SP600125 (SP) or with vehicle and then incubated for 18 h in medium containing 25 μg/ml fucoidan alone (Fuc), 0.5 μM thapsigargin (Tg) alone, or both compounds. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (C) WT or Jnk2 ^−/− macrophages were incubated for 18 h in medium alone or medium containing ac-LDL plus 58035, 0.5 μM thapsigargin, or 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Quantitative apoptosis data for each condition are shown as described in (A). Data are expressed as mean ± SEM (n = 4). (D) Macrophages were incubated with 0.5 μM thapsigargin or 0.5 μM thapsigargin plus 25 μg/ml fucoidan for 0, 1, 2, and 3 h. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr ¹⁸³/Tyr¹⁸⁵ JNK (phospho-JNK, top panel) and total JNK (bottom panel). (E) Macrophages were preincubated for 30 min with medium alone or medium containing the anti-SRA antibody 2f8 or isotype control IgG2b (30 μg/ml). The cells were incubated for 3 h in medium alone or medium containing 0.5 μM thapsigargin plus 25 μg/ml fucoidan. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for activated phospho-Thr ¹⁸³/Tyr¹⁸⁵ JNK (top panel), and total JNK (bottom panel). (F) WT and Jnk2 ^{− / −} macrophages (Mφs) were FC loaded for 0 or 12, and 15 h using 100 μg/ml ac-LDL plus the ACAT inhibitor 58035. Whole cell lysates were prepared as described in “Materials and methods,” and were immunoblotted for CHOP (top panel), total JNK (middle panel), and actin (bottom panel).