Supplemental Figure Legends
Figure 1. Results obtained from the treated cells reflect the effects of the drugs and
not of DMSO. (A) Cells were treated with the indicated concentration of DMSO for 48 h. The
percentage of viable cells was determined by DIMSCAN analysis. Values represent the
percentages of cell viability normalized to that of the untreated cells and are the mean of
three separate treatments. (B) Western blot analysis of phospho-H2A.X was performed on
the lysates from cells treated for 24 h with the indicated concentration of DMSO. The levels
of β-actin protein served as loading controls. Results are representative of three independent
experiments. As positive control we loaded the protein lysate from Ly3 cells treated with 10
M SRT2183, in which the final concentration of DMSO is 0.125%. (C) TaqMan real-time
PCR was performed on the cDNA from cells treated for 24 h with the indicated concentration
of DMSO. The mRNA levels were normalized to levels of β-actin mRNA. Values represented
as bar graphs are the mean of 3 independent experiments with the standard deviation. Data
are expressed as 2^ddCt. Positive control= Ly3 cells treated with 10 M SRT2183 + 10 nM
panobinostat, in which the final concentration of DMSO is 0.135%.
Figure 2. SIRT1 activators inhibit STAT3 signaling and reduce c-Myc protein levels in
Reh cells. Reh cells were treated with the indicated concentrations of SRT501 or SRT2183
for 24 h. Following this, cells were analyzed by flow cytometry for total- (Ai, Bi) and phosphoSTAT3 (Aii, Bii) protein levels. (C) Western blot analysis of c-Myc was performed on the
lysates from Reh cells 24 hours post-treatment. The levels of β-actin protein served as
loading controls. Results are representative of three independent experiments. MFI: median
fluorescence intensity.
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Figure 3. Combination of SRT2183 with panobinostat induces greater reduction of cMyc, phosphorylation of H2A.X, and acetylation of p53 and H4 in ALL cell lines. Reh
cells (A), NALM-6 cells (B) and MOLT-4 cells (C) were treated for 24 h with the indicated
concentrations of SRT2183, panobinostat or a combination of these drugs. Following this,
Western blot analysis of c-Myc, acetyl-p53, phospho-H2A.X and acetyl-H4 proteins was
performed on the lysates from those cells. The levels of β-actin protein served as loading
controls. Results are representative of three independent experiments.
Figure 4. GADD45G gene map. The oligos for EMSA are highlighted in blue and yellow; the
yellow portion of each oligo corresponds to the NF-kB consensus binding sequences. The
primers for ChIP assay are shown as boxes with solid lines. The red fonts represent the
coding sequences of GADD45G gene.
Table 1. SRT2183 upregulates expression of DNA damage and apoptosis related genes
in Reh cells. Following treatment with 10 uM SRT2183 for 24 hours, PCR array analysis was
performed on Reh cells. The values represent the gene upregulation folds, which were
calculated by the ratios of treated versus control cells relative gene expression, normalized to
the levels of β-actin mRNA.
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