U937 cells were incubated with or without FK866 at the indicated concentrations for 48 h. with or without EX527, cambinol, BU, or VA at the indicated concentrations. Viability was assessed 48 h later by PI cell staining and circulation cytometry. CI values refer to the highest drug concentrations used.(PDF) pone.0022739.s003.pdf (39K) GUID:?865B70E2-5AFA-4299-A373-9ADCD35A20E2 Physique S4: Sirtuin inhibitors and HDAC inhibitors show poor activity and fail to cooperate in healthy PBMCs. ACC, Healthy PMBCs were incubated in 96-well plates with or without sirtinol, cambinol, BU, or VA at the indicated concentrations. Viability was assessed FLNB 48 h later by PI cell staining and circulation cytometry. CI values refer to the highest drug concentrations used. Results are means SD of three impartial experiments with three different donors.(PDF) pone.0022739.s004.pdf (33K) GUID:?5DDFDBE2-94E9-4C36-88B9-3C7A78DEF4B6 Physique S5: SIRT1 silencing enhances HDAC inhibitor activity in Jurkat cells. A, B. Jurkat cells were transfected with a non-targeting siRNA (cntr siRNA) or with an anti-SIRT1-siRNA. Thereafter, two days later, cells were utilized for protein lysate preparation or plated in 96-well plates for viability assays. A, SIRT1 and -tubulin levels were determined by immunoblotting. B, Cells were incubated in the presence or absence of the indicated concentrations of VA or of BU. Two days later, lifeless cells were quantified by PI staining and circulation cytometry. One representative experiment out of three is usually offered. *: p<0.05.(PDF) pone.0022739.s005.pdf (68K) GUID:?A7F416B1-359D-44A1-9388-B8A418EB82E8 Figure S6: SIRT1 expression in primary leukemia cells and in leukemia cell lines. A, RNA was extracted from freshly isolated normal PBMCs (n?=?10), main B-CLL cells (n?=?36), AML cells (n?=?11), and from your cell lines U937, Jurkat, and 697. SIRT1 levels were determined by Q-PCR. SIRT1 levels in main leukemia samples (A) and in cell lines (B) were compared to the imply value obtained in PBMCs using the 2 2?CT method.(PDF) pone.0022739.s006.pdf (92K) GUID:?6DBB6AA5-4758-4287-A5F0-651F6EC64CDE Physique S7: Correlation between SIRT1 expression and activity of the combination sirtuin/HDAC inhibitors in leukemia cells. Main B-CLL cells were plated in 96 well plates and treated with or without 100 g/ml VA, 500 M BU, 50 M cambinol, or their combinations. Dead cells were enumerated two days later by PI staining and circulation cytometry. The correlation between the CI (A, B) or the overall cytotoxic activity (C, D) of each drug combination and SIRT1 expression was assessed by Pearson correlation coefficient.(PDF) pone.0022739.s007.pdf (98K) GUID:?BFAF4B97-BA11-46F6-A2C9-7A51DEDE5DCF Physique S8: Correlation between SIRT1 expression and activity of the combination FK866/HDAC inhibitors in leukemia cells. Main B-CLL cells were treated with or without 10 nM FK866, 100 g/ml VA, 500 M BU, or their combination. Dead cells were enumerated four days later by PI staining and flow cytometry. TLR7-agonist-1 The correlation between the CI (A, B) or the overall TLR7-agonist-1 cytotoxic activity (C, D) of each drug combination and SIRT1 expression was assessed by Pearson correlation coefficient.(PDF) pone.0022739.s008.pdf (99K) GUID:?4FDCD545-70F9-4B09-B7D7-B4C81D8C17F4 Figure S9: zVAD-fmk reduces cell death in response to sirtuin and HDAC inhibitors in leukemia cells. A, Jurkat cells were pre-incubated for 1 h with or without 100 M zVAD-fmk. Thereafter, 100 g/ml VA, 30 M sirtinol, 75 M EX527, or their combinations were added as indicated. Viability was assessed two days later by PI staining and flow cytometry. BCD, 697 cells were pre-incubated for 1 h with or without 100 M zVAD-fmk. Thereafter, TLR7-agonist-1 100 g/ml VA, 30 M sirtinol, or their combination were added as indicated. Two days later, cells were imaged by light microscopy (D), dead cells were enumerated by PI staining and flow cytometry (B), and hypodiploid cell nuclei were counted by PI staining of isolated cell nuclei and flow cytometry (C).(PDF) pone.0022739.s009.pdf (483K) GUID:?1801D714-6C1D-4945-A286-6D898C01A73A Figure S10: VA induces Bax upregulation in leukemia cell lines, but not in healthy PBMCs..