FIGURE 3.

ATF4 mediates fenretinide- and bortezomib-induced cell death. A and B, SH-SY5Y and A375 cells were transfected with siRNAs for ATF4 or with a non-silencing control siRNA (ctrl) prior to treatment with fenretinide (FenR) (SH-SY5Y, 5 μm; and A375, 10 μm), bortezomib (Bort) (SH-SY5Y, 5 nm; and A375, 30 nm), or thapsigargin (Thap) (SH-SY5Y, 1.5 μm; and A375, 7.5 μm) for 6 h. ATF4 or Noxa mRNA was measured by real-time PCR relative to β-actin as an internal control (A). ATF4 (lower band indicated by the asterisk in SH-SY5Y cells), ATF3, Noxa, and β-actin expression was determined by Western blotting (B). C, SH-SY5Y and A375 cells were transfected with siRNAs for ATF4 or with a non-silencing control siRNA prior to treatment with fenretinide (SH-SY5Y, 10 μm; and A375, 15 μm), bortezomib (SH-SY5Y, 5 nm; and A375, 50 nm), or thapsigargin (SH-SY5Y, 3 μm; and A375, 10 μm) for 24 h. Apoptosis was measured by flow cytometry of propidium iodide-stained cells to determine the sub-G₁ fraction. Data are expressed as the percentage total population or relative to control untreated cells; each point is the mean ± S.E. (n ≥ 3). D, A375 cells were treated with fenretinide (15 μm), bortezomib (50 nm), or thapsigargin (10 μm) for 6 h. Recruitment to the Noxa promoter was determined by promoter pulldown assays in the absence (control) or presence of the Noxa promoter DNA fragment, followed by Western blotting for ATF4. Data are shown for whole cell extracts; similar results were obtained with nuclear extracts (not shown).