Figure 1
(A). TSA induces Akt and GSK3β dephosphorylation in MCF-7 breast cancer cells. MCF-7 cells were incubated with 1 μM TSA for the indicated times. Following incubation, the cells were harvested and lysates were resolved by SDS-PAGE. Proteins were detected using the indicated antibodies. (B). The relative amounts of pAkt and pGSK3β in A were measured by densitometry and normalised to the amount of p38/SAPK. Result is representative of at least three separate experiments. (C). Knockdown of class I HDAC proteins induces Akt and GSK3β dephosphorylation. MCF-7 cells were transfected with oligo pools specifically targeting HDAC1, 2, 3 or a non-targeting siRNA pool (NSC). 72 h after transfection, cells were harvested and lysed. Lysates were treated as in A and probed with the indicated antibodies. (D). siRNA-mediated GSK3β knockdown attenuates the cytotoxic effect of TSA on MCF-7 cells. MCF-7 cells were transfected with oligo pools specifically targeting GSK3β or a non-targeting siRNA pool (NSC). 24 h after transfection cells were harvested and reseeded in 96-well plates and incubated for 24 h. Cells were then treated with 1 μM TSA for 48 h and relative cell survival was measured as described in materials and methods. Results represent the mean ± S.E. from at least three separate experiments. * P < 0.001 TSA treated vs. untreated NSC siRNA cells, ** P < 0.001 TSA treated NSC vs. TSA treated GSK3β siRNA cells. Inset: Lysates from cells transfected in parallel were probed with antibodies directed against GSK3 to monitor siRNA efficiency. (E). Effect of GSK3β siRNA on TSA induced cytotoxicity. Cells were treated as in D and examined by flow cytometry (see materials and methods section). Result is representative of at least three separate experiments. (F). Effect of GSK3β inhibition on TSA induced cytotoxicity. MCF-7 cells were cultured in 96-well plates with 10-9 – 10-5 M TSA alone or in combination with 10 mM LiCl. Relative cell survival was determined after 48 h as described in materials and methods section. Result is representative of three separate experiments.