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Fig. S1 (A) DAPI counterstaining of fat tissue SA-βGal staining described in Figure 1D, showing cytoplasmic localization of the βGal signal. (B) Effect of DBC1 knockdown on apoptosis triggered by H2O2 in 3t3-L1 preadipocytes. Cells were incubated with 200 μm H2O2 for 2 h, washed and let them recover for 4 more hours. Apoptosis was determined by nuclear shape using DAPI as nuclear marker. Pictures were taken blindly before and after treatment and apoptosis was independently evaluated by counting cells in the field based in nuclear shape, size, and DNA condensation. Results shown represent average ± SEM of 3 independent experiments. (C) Western blot for DBC1, HDAC3, and SIRT1 in H2O2–treated 3T3-L1 preadipocytes transfected with the different siRNAs and collected at the time of H2O2 treatment. (D) Densitometry analysis for p21 expression in three independent experiments corresponding to the results shown in Figure 2E. (E) Quantitation of the effect of DBC1, SIRT1, and HDAC3 siRNA on γ-H2.AX foci in 3T3-L1 preadipocytes after incubation with H2O2 (200 μm) shown in Figure 2F. Connecting lines show significant differences between conditions (< 0.05, ANOVA, n = 3). (F) Chromatin immunoprecipitation (ChIP) for the p21 and p16 promoter regions in 3T3-L1 preadipocytes using an antibody against HDAC3. Nonspecific IgG was used as control. The results shown are the average ± SEM of 4 independent ChIP. (*P < 0.01; t-test).

Data. S1 Methods.

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