Figure 2.

NH125 treatment leads to key ISR signaling events culminating in CHOP-mediated DR5 TRAIL synergy. A, U251 cells were treated with increasing concentrations of NH125 for 24 hours and probed for ISR signaling events. Incubating U251 cells with increasing concentrations of NH125 produces an increase in EIF2α phosphorylation accompanied by an increase in ATF4, CHOP, and DR5 expression. B, In a parallel set of experiments, lysate from U251 incubated with 2.5 μmol/L NH125 for 0,2,4,8,12, and 24 hours was probed for ISR signaling events. EIF2a phosphorylation, ATF4, CHOP, and DR5 expression occur as early as 2 hours. C, EIF2a phosphorylation, ATF4, CHOP, and DR5 expression are observed across a panel of glioma and non-small cell lung cancer cells treated with 5 mmol/L NH125 for 24 hours. This pattern is also observed in U251 cells treated with 1 μg/mL of Tunicamycin for24 hours. D, U251 cells were treated with increasing concentrations of NH125 for 24, and then co-incubated with either 25 ng/mL or 100 ng/mL of TRAIL for 4 hours. Raw viability data were normalized to 0.1% DMSO-treated U251, and means and SDs from biological triplicates were plotted. Addition of 25 or 100 ng/mL of TRAIL produces a noticeable decrease in cell viability (*** indicates combination index <0.5, see Supplementary Table S1). Following successful knockout of CHOP, U251 cells were treated with increasing concentrations of NH125 for 20 hours, and then co-incubated with either 25 or 100 ng/mL of TRAIL for 4 hours. Raw viability data were normalized to 0.1% DMSO-treated CHOP knockout cells, and means and SDs from biological triplicates were plotted. Knockout of CHOP led to an abrogation of TRAIL-mediated cell death at both 25 and 100 ng/mL. E, Knockout of CHOP leads a dramatic reduction in DR5 expression following treatment with 2.5 and 5.0 μmol/L NH125 for 24 hours. Similarly, CHOP knockout cells treated with 1 μg/mL Tunicamycin for 24 hours also display decreased DR5 expression.