FIG. 6.

CM from APAP-treated macrophages decreased SIRT1 protein levels in hepatocytes. (A) Hepatocytes were treated 16 h with conditioned media collected from RAW 264.7 cells treated with vehicle or 5 mM APAP for 8 h and then reefed with fresh medium for a further 16 h (control-CM or APAP-CM, respectively). Representative immunoblots showing SIRT1 and α-Tubulin (loading control) protein levels (left). After quantification of all blots, results are expressed as fold change relative to control-CM condition (right). Values are mean ± SEM. ***p < 0.001 versus control-CM according to Student's t-test. (B) Hepatocytes were treated with APAP (0.5 and 1 mM) for 16 h. Representative immunoblot showing SIRT1 and α-Tubulin (loading control) protein levels (left). After quantification of all blots, results are expressed as fold change relative to vehicle condition (right). Values are mean ± SEM. (C) Hepatocytes were treated with control-CM or APAP-CM for 16 h, as indicated (A). PGC1α was immunoprecipitated with anti-PGC1α antibody or protein A agarose as a negative control. Immunoblot against anti-Acetil-Lys represents acetylated PGC1α protein levels. Total α-Tubulin was analyzed in the SUP-IP as a loading protein control. (D) Hepatocytes were treated with APAP-CM for the indicated times in the presence of the proteasome inhibitor MG132 (10 μM) and then the levels of ubiquitinated SIRT1 were analyzed by immunoprecipitation (upper blot). Total SIRT1 protein detected in the WCL is shown in the lower blot. APAP-CM, conditioned medium of APAP-treated macrophages; PGC1α, peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha; SUP-IP, IP-supernatant; WCL, whole-cell lysate.