MT-4 regulated HSP27 protein expression.

<p>(A) HSP27, HSP70, and HSP90 mRNAs were analyzed from the Bonome dataset. (B) A2780 and NCI-ADR/res cells were lysed and analyzed by western blot for HSP27 protein. Lane 1, A2780 and Lane 2, NCI-ADR/res. (C) NCI-ADR-res cells were treated with the indicated concentrations of MT-4 (0.05 to 3 μM) and DMSO (Control, C) for 48 h. (D) NCI-ADR/res cells were treated with 0.3 μM MT-4, 10 μM paclitaxel (T), or 10 μM vincristine (Vin) or DMSO (Control, C) for 48 h. (E) NCI-ADR/res cells were treated with 0.3 μM MT-4, SB203580 alone (20 μM or 50 μM) or a combination of both reagents for 48 h. Whole cell lysates were evaluated using western blot analysis for the indicated proteins. (F) NCI-ADR/res cells were transfected with or without 2 μg of domain-negative p38 plasmid and then incubated with MT-4 (0.3 μM) for 48 h. Whole cell lysates were subjected to western blot analysis. Similar results were obtained in at least three other independent experiments. (G) Co-immunoprecipitation (Co-IP) assay and western blot assay. NCI-ADR/res cells were treated as indicated, and lysates were subjected to a Co-IP assay with anti-caspase-3 and were blotted with anti-HSP27 and anti-caspase-3. (H) The cells were transfected with or without 5 μg of wild-type HSP27 plasmid and then incubated with MT-4 (0.3 μM) for 48 h. Whole cell lysates were subjected to western blot analysis and (I) cell death detection kit assays. *<i>p</i> < 0.05. The data represent the mean ± S.E.M. of at least three independent experiments. Densitometric analysis shows fold change in protein levels compared with control group by image J.