Figure 7.

Active benzodiazepinone 4hh, but not inactive compound 4ll, inhibits calcium regulator-induced p38 MAPK activation. H4-CHOP-luciferase reporter cells were pretreated with DMSO or compounds for 1 h and then treated with thapsigargin (20 nM), CDDO-IM (600 nM), ionomycin (120 nM), or MG132 (740 nM) for an additional 18 h. The luciferase expression was assessed using the Steady-Glo luciferase assay reagent. The p38 inhibitor was used at 20 μM. The benzodiazepiones 4hh or 4ll (10 μM) were used with thapsigargin or CDDO-IM treatment, while concentrations of 20 and 50 μM were tested with MG132 and ionomycin treatment, respectively. Wells treated with compounds only but no inducers were used as background control correspondingly, and wells treated with inducers but no compounds were used as 100% controls. The RLU (% of control) = 100 × (well value – average of background)/(average of 100% control – average of background). The data were analyzed using MS Excel software. The experiments were repeated at least three times, and the data are represented as the average ± SEM. Statistical significance (P < 0.05) was determined using one-way analysis of variance. *Significance was compared with inducer treatment groups.