Figure 2. Hsp90 reversed luteolin-induced phosphor-STAT3 and Akt down-regulation.

A, HeLa cells were transfected with 0.5, 1, 1.5, 2 µg HA-Hsp90 plasmids and incubated with (+) or without (−) 50 µM luteolin for 24 h before harvesting. Western blotting with specific antibodies was performed for p-STAT3, STAT3, and Akt respectively. B, HeLa cells were transfected with HA-Hsp90 (2 µg) or empty vectors for 24 h, and then cells were treated with 50 µM luteolin for 24 h. The endogenous Akt, STAT3 and Tyr⁷⁰⁵-phosphorylated-STAT3 were measured by immunoblot analysis respectively. C, HeLa cells were transfected with pSuper-Hsp90i or pSuper vector, and then were subjected to immuoblot analysis for measuring indicated proteins. D. As a control, HeLa cells were transfected with Hsp70 nonsense or antisense oligonucleotides, and then were analyzed by western blotting using anti-p-STAT3, anti-STAT3, anti-Akt antibodies. E, HeLa cells were first transfected with pSuper-Hsp90i or pSuper, and 24 h later, cells were transfected with HA-Hsp90 or vector. And then cells were treated with luteolin for 24 h. Lysates were subjected to immunoblotting for testing p-STAT3, STAT3, Akt, Hsp90. F, HeLa cells were first transfected with pSuper-Hsp90i and then transfected with HA-Hsp70. After 24 h cells were treated with luteolin for 24 h, and subjected to immunoblotting for detecting indicated protein levels.