Myc activation increases global translation, stimulates BZ-mediated aggresome formation, and sensitizes HFFs to BZ + SAHA–induced apoptosis. (A) Activation of Myc increases the expression of the target genes eIF4E and HDAC6. HFFs expressing the MSCV-IRES-puro or MSCV-Myc-ER^TAM-IRES-puro retroviruses were treated with 1 μM 4-HT for 24 hours. Lysates were prepared and immunoblotting was performed as described in “Immunoblot analyses.” (B) Myc activation increases global translation rates of HFFs. HFFs expressing the MSCV-IRES-puro or MSCV-Myc-ER^TAM-IRES-puro retroviruses were treated with 1 μM 4-HT for 24 hours and equal cell numbers were then incubated with [³⁵S]cysteine/[³⁵S]methionine for 30 minutes. Protein was collected as described in “Metabolic labeling” and [³⁵S]cysteine/[³⁵S]methionine incorporation was measured using a scintillation counter. Mean (n = 3), SD. * indicates a significant difference from MSCV-IRES-puro–transfected cells, P < .05. (C) Myc activation stimulates aggresome formation in HFFs treated with BZ. HFFs expressing the MSCV-IRES-puro or MSCV-Myc-ER^TAM-IRES-puro retroviruses were plated on chamber slides and pretreated with 1 μM 4-HT for 24 hours and then treated with 100 nM BZ, 2 μM SAHA, or both agents. After treatment, cells were fixed and stained and images were obtained by confocal microscopy. Magnification = 40×. (D) Quantification of aggresome formation. Approximately 200 cells were counted and scored as aggresome positive or negative. Mean (n = 3), SD. * indicates a significant difference from bortezomib-treated cells, P < .05. (E) Cycloheximide blocks BZ- and BZ + SAHA–induced apoptosis in Myc-expressing HFFs. The indicated cells were pretreated with 1 μM 4-HT and were then treated with 100 nM BZ, 2 μM SAHA, or both in the presence or absence of 50 μM cycloheximide for 24 hours. Apoptosis was measured by PI-FACS analysis. Mean (n = 3), SD. * indicates a significant difference from cells treated without cycloheximide, P < .05.