Figure 4

The effects of some signaling inhibitors on proliferation inhibition and HSP70 induction-related molecules activations. (A) Isobologram analysis for combined effects of celastrol and the inhibitors reducing celastrol’s HSP70 induction on proliferation inhibition. U937 cells were incubated with different doses of celastrol or the inhibitors for 24 h, and the numbers of vital, dead, and total cells were counted by FCM. D_m value (the median-effect dose or concentration) of each drug was obtained using Calcusyn 2.0 software. According to the D_m value, various concentrations of single agents and agent combinations with a fixed constant ratio were tested. The isobologram analysis was analyzed with Calcusyn 2.0 software. Combination index (CI) = 1 refers to additive effect, CI < 1 refers to synergism, and CI > 1 means antagonism. ED50, ED75, and ED90 represent effective doses of 50%, 75%, and 90% inhibition, respectively. The values were shown as mean ± SD. * P < 0.05 vs. 1. (B) Effects of inhibitors reducing celastrol’s HSP70 induction on the HSP70 regulation signaling pathway. U937 cells were pre-incubated with the inhibitors for 1 h, followed by treatment with 600 nM celastrol for 10 min. Total proteins were extracted by lysis buffer with phosphorylation inhibitor. WB assayed phosphorylated or total signaling molecules.