Figure 2. HDAC6 requirement for TGF-β1-activation of Notch Signaling in A549 cells.

(A) Serum-starved A549 variants were exposed to TGF-β1 (2.5 ng/ml) for the indicated duration, cell lysates were fractionated to cytoplasmic and nuclear extracts and protein levels of nuclear ICN and HDAC6 were examined by Western analysis. (B) RNA was isolated from A549 variants exposed to TGF-β1 (2.5 ng/ml) for 24 hours. Quantitative RT-PCR was carried out for HEY-1. (C) Same experimental conditions as in B, except the transcript of interest examined was HES-1. The –fold change of each transcript was obtained by setting the value of the unexposed pS cells to 1 (D) Experimental conditions and fractionation of serum starved A549 variant cell lysates were the same as in A, except the duration of TGF-β1 treatment was for 24 and 48 hours. Protein levels of HEY-1 and HES-1 in the nuclear extract were examined by western analysis. (E) Cytoplasmic fraction of the same experiment as D, except TGF-β1 responsive genes PAI-1 and Vimentin protein levels were examined by western analysis. (F) Densitometry analysis of western blots from three independent experiments represented in panel (D); levels of HEY-1 and HES-1 were relativized to lamin A/C. For qPCR analysis data presented as mean +/− SEM of triplicate wells and are representative of three independent experiments statistically analyzed using one-way ANOVA. For densitometry analysis data presented as mean +/− STD of three independent experiments statistically analyzed using one-way ANOVA. *P <0.05, **P < 0.01, and ***P < 0.001 compared with the relative control.