Figure 3

The HSF-1–HSP70–STAT1 signaling pathway is involved in bortezomib (BTZ)-induced cytotoxicity. (a) TOV112D cells were treated with 0.05 μM of BTZ for 24 h. HSP70 mRNA and protein levels were measured by real-time quantitative PCR and western blot analysis, respectively. (b) The effects of the HSP70 knockdown by short hairpin (sh)RNA and the treatment with 0.05 μM of BTZ for 24 h in TOV112D cells were analyzed by measuring the levels of caspase-3 cleavage and (c) using the LDH assay. For clarity of presentation, data were normalized with that of BTZ-treated cells, which was set as 1. (d) The effects of the HSP70 knockdown by shRNA on the regulation of STAT1 phosphorylation in BTZ-treated TOV112D cells were determined by immunoblotting. (e) Treatment with BTZ stimulated phosphorylation of STAT1 at tyrosine 701 and serine 727, which was enhanced by overexpression of HSP70. (f) The overexpression of HSP70 attenuated BTZ-mediated growth inhibition. Cell viability was analyzed using the MTT assay. (g) TOV112D cells were transfected with an HSE reporter construct. The dose-dependent activation of HSF-1 was measured using luciferase assays after 24 h of treatment with BTZ. (h) TOV112D cells were treated with 0.05 μM of BTZ for 24 h, and the effects of HSF-1 knockdown on the regulation of HSP70 and caspase-3 cleavage were analyzed by immunoblotting. For clarity of presentation, data were normalized with that of BTZ-treated cells, which was set as 1. Data are expressed as mean±standard error of the mean. The results are representative of at least three independent experiments