Effects of transient SIRT1 knockdown on expression of SASP components.

<p><b>A</b>, MRC-5 cells were transfected with siRNA against SIRT1 (siSIRT1–8 and siSIRT1–9) or control (siControl), and then lysed 2 days after transfection. Cell lysates were subjected to immunoblot analysis using antibodies to SIRT1 and <b>γ</b>-tubulin (loading control). <b>B</b>, MRC-5 cells were transfected as in A and cultured for 2 days. The resulting cells were exposed (or not) to 10Gy of X-radiation. Cells were lysed on the indicated days after irradiation and subjected to immunoblot analysis using antibodies to IL-8, IL-6, and γ-tubulin. <b>γ</b>-Tubulin was used as a loading control. <b>C</b>, SIRT1 knockdown and control cells were exposed (or not) to 10Gy of X-irradiation. Total RNA was extracted on the indicated days after irradiation. IL-8, IL-6, IL-1β, and GRO-α mRNA levels were then determined by quantitative RT-PCR analysis. The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results.