FIG 1.
PEITC is a robust inducer of the heat shock response. (A) Chemical structures of allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC). (B and C) MDA-MB-231 cells (2.5 × 105 per well) (B) or mouse embryonic fibroblasts (MEF, 2 × 105 per well) (C) growing in six-well plates were exposed to vehicle (0.1% acetonitrile), AITC, BITC, or PEITC for 16 h. Cells were lysed in RIPA buffer, and proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and probed with an antibody against Hsp70. The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. (D) Wild-type MEF cells (2 × 105 per well) in six-well plates were exposed to vehicle (0.1% acetonitrile) or PEITC for 8 or 16 h. The cells were lysed, and the total RNA was extracted and reverse transcribed into cDNA. The mRNA levels for hspa1a were quantified using real-time PCR. The data were normalized using β-actin as an internal control. (E) MDA-MB-231 cells (2 × 106 per well) growing in 10-cm dishes were exposed to vehicle (0.1% acetonitrile) or 20 μM PEITC for 3 h. The cells were then fixed with 0.4% (wt/vol) paraformaldehyde. Cell lysates were subjected to nuclear (N) and cytoplasmic (C) separation, and proteins were resolved by SDS-PAGE (10% gel), transferred to Immobilon-P membranes, and probed with an antibody against HSF1. The levels of lamin B2 and GAPDH served as fraction purity indicators and as loading controls. (F) HeLa-HSE-luc cells (1.3 × 105 per well) stably transfected with the luciferase gene under the control of the HSP70.1 promoter were grown in 12-well plates and exposed to vehicle (0.1% acetonitrile) or PEITC. The luciferase activity was determined in cell lysates. The relative luminescence units (RLU) were quantified and normalized with respect to the vehicle control treatment. Data represent means ± the SD and are expressed as the ratio of the relative transcripts in treated to the control samples.