Senescent WI-38 cells are resistant to the effects of HDAC inhibition. (A) Butyrate and TSA did not further enhance the expression of cell cycle inhibitor protein p21WAF1 in senescent WI-38 cells. Proliferating and senescent WI-38 cells were treated with butyrate (BA) or TSA for 24 hours, as indicated. Cytosolic extracts were prepared and equivalent protein levels were analyzed by immunoblot using an antibody specific for p21WAF1. Actin served as a loading control and was determined by immunoblotting for the actin protein. Control samples (0 hours) consisted of cytosolic extracts prepared from untreated cells. (B) The effect of HDAC inhibitors on proteasome activity in proliferating and senescent WI-38 cells. Cytoslic extracts were prepared from proliferating (Young;◆) and senescent (Old;) WI-38 cells treated with butyrate (+ Butyrate) or TSA (+ TSA) for 0, 24, 48 and 72 hours, as indicated. Equivalent protein concentrations were determined and analyzed for proteasome activity using a synthetic fluorogenic substrate (± standard error; n = 4; values of non-treated samples set to 1). Senescent cells were found to be significantly less responsive to proteasome inhibition by butyrate and TSA.