Figure 2. 147-dependent ATF6 activation is sensitive to increases in cellular free thiols and resveratrol.

(A) Illustration showing a mechanism whereby 147 is converted to an o-quinone imine (147-QI) followed by tautomerization to a quinone methide (147-QM), which can then react with cellular proteins through nucleophiles such as free Cys side chains. (B) Bar graph showing the activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated with 147 (10 µM) and/or β-mercaptoethanol (BME; 55 μM or 110 μM) for 18 hr. Error bars show SEM for 3 independent experiments. *p<0.05. (C) Bar graph showing the activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated with thapsigargin (Tg; 0.5 µM) and/or β-mercaptoethanol (BME; 55 μM or 110 μM) for 18 hr. Error bars show SEM for 3 independent experiments. (D) Bar graph showing the activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated with 147 (10 µM) and/or resveratrol (10 µM) for 18 hr. Error bars show SEM for 4 independent experiments. ***p<0.005. (E) Bar graph showing the activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated with Tg (500 nM) and/or resveratrol (10 µM) for 18 hr. Error bars show SEM for 4 independent experiments.

Figure 2—figure supplement 1. 147-dependent ATF6 activation is sensitive to increases in cellular free thiols and resveratrol.

(A) Structure of Eugenol, which can be metabolically oxidized to create a p-quinone methide similar to that predicted to be formed in the mechanism of 147-dependent ATF6 activation (see Figure 2A). (B) Structure of acetaminophen, which can be metabolically activated to an N-acetyl-p-benzoquinone imine, similar to that which may form in the oxidation of 147 (see Figure 2A). (C) Bar graph showing the activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated with 147 (10 µM), thapsigargin (Tg; 0.5 µM) and/or N-acetyl cysteine (NAC; 5 mM), added 4 hr prior to addition of 147 or Tg, for 18 hr. Error bars show SEM for three technical replicates.