Figure 4. Doxorubicin-induced apoptosis in senescent cells.. (A) Senescent WI-38 cells are resistant to doxorubicin-induced apoptosis. Early passage and senescent cells were incubated in the presence of 300 nM doxorubicin for 72 hours and the fraction of apoptotic cells was determined by the Annexin V assay. (B) Western blot analysis of early passage and senescent WI-38 cells treated with 300 nM doxorubicin for 24 hours. (C) Transcriptional activity of p53 target genes in doxorubicin-treated senescent WI-38 cells. Early passage and senescent cells were exposed to 300 nM doxorubicin, RNA was extracted for and data analyzed as in Figure 3B. (D) Effect of doxorubicin on the relative expression levels of p53 target genes in senescent cells. Cells were treated as in (C) and data calculated and presented as in Figure 3C. (E) Nutlin raises doxorubicin-induced p53 protein level in senescent cells. Early passage and senescent WI-38 cells were exposed to 300 nM doxorubicin, 10 μM Nutlin-3a, or combination of both for 24 hours prior to collection for Western analysis. (F) Nutlin increases apoptosis induced by doxorubicin in senescent cells. Senescent WI-38 cells were treated with 10 μM nutlin-3a, 300 nM doxorubicin or 10 μM nutlin-3a plus 300 nM doxorubicin for 72 hours and the apoptotic cell fractions were measured by the Annexin-V assay. (G) Nutlin restores transcriptional response to doxorubicin-induced p53 in senescent cells. Senescent cells were treated with 10 μM nutlin-3a, 300 nM doxorubicin or 10 μM nutlin-3a plus 300 nM doxorubicin for 24 hours and expression levels of indicated genes were determined by quantitative PCR, normalized, and calculated as fold change. (H) Nutlin restores the transcription of doxorubicin-induced p53 target genes in senescent cells to early passage levels. Early passage cells were exposed to 300 nM doxorubicin and senescent cells to 300 nM doxorubicin plus 10 μM nutlin-3a. 24 hours after treatment, mRNA levels were determined by quantitative PCR and normalized to expression levels in early passage cells (100%).