Supplementary Figure melatonin does not induce apoptosis or senescence. (A)
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| Supplementary Figure S1. Melatonin does not induce apoptosis or senescence. (A) HFF cells were grown for the indicated time in the absence and in the presence of 1µM melatonin; cells were counted and cell number is shown in the graph. (B) MCF-7 and HCT 116 cells were treated with melatonin for the indicated time and BrdU incorporation was performed. Cell cycle distribution is shown. (C) MCF-7 and HCT 116 cells were treated with melatonin for the indicated time cell cycle distribution was analysed. (D) MCF-7 and HCT 116 cells were treated with melatonin or CDDP for the indicated time and Annexin V staining was performed. Percentage of Annexin V positive cells is shown. (E) MCF-7 and HCT 116 cells were treated with melatonin for the indicated time. Cells were fixed and SA-β-gal staining was performed. WRN -/- fibroblasts (G) were used as a positive control. Arrows indicate senescent cells. (F) MCF-7 and HCT 116 cells were treated with melatonin for the indicated time. Cell lysates underwent immunoblot. The ratio between Phospho-p53(Ser15) and p53 is shown in the graph.
Supplementary Figure S2. Melatonin does not counteract apoptosis induced by chemotherapy. MCF-7 cells were treated as in Figure 2D; 5·102 cells were seeded in 35mm dishes and allowed to grow for 15 days. Percentage of colonies relative to control is shown. Supplementary Figure S3. Inhibition of cell growth by melatonin is p53-dependent. Stable MCF-7 sh-LacZ (A) and sh-p53 (B) cells, HCT 116 (C) and HCT 116 p53-/- (D) were grown for the indicated time with and without melatonin in the absence and in the presence of CDDP. Number of cells/ml is shown. Supplementary Figure S4. Melatonin induces H2AX phosphorylation. (A-D) MCF-7 (A) and HCT 116 (C) were treated with melatonin for the indicated time and cell lysates underwent immunoblot with the indicated antibodies. Panels B and D show densitometryc quantification of phosphorylated and total H2AX signals. (E-F) MCF-7 (E) and HCT 116 (F) cells were treated with melatonin and/or CDDP for the indicated time and underwent immunofluorescence with the indicated antibodies. Supplementary Figure S5. (A-B) MCF-7 (A) and HCT 116 (B) cells were treated with melatonin for the indicated time and underwent immunofluorescence with the indicated antibodies. Supplementary Figure S6. Chemical inhibition of p38 MAPK impairs p53 phosphorylation upon melatonin. Cells were treated as in Figure 5B and 6B. Densitometric analyses of p53 phosphorylated at Ser-15 in MCF-7 (A) is shown. Two-tails t-test was performed on 3 independent experiments. Error bars indicate standard deviations. |