Table S1. New colony formation 7 days after stimulation with doxo and VCR in JURKAT cells

Transcription

1 Table S1. New colony formation 7 days after stimulation with and in JURKAT cells drug co + number of colonies 7±14 4±7 48±11 JURKAT cells were stimulated and analyzed as in Table 1. Drug concentrations were reduced compared to the experimental setting in Supplemental Figure 4 according to lower cell density. Data are presented as mean of 3 independent experiments ± SEM. p<,5, comparing new colony formation after alone to the combined plus treatment.

2 Figure S1. Determination of apoptotic cell death by / double staining CEM cells were stimulated as in Fig. 1A and cell death induction determined by / propidium iodide double staining (left panels). In parallel, forward side scatter analysis (right panels) was performed for each stimulatory setting for 36 (upper panel) and 48 (lower panel) hours incubation time. Figure S2. Signaling mechanism of and inhibition by (A) Western blot of total cellular protein of CEM cells was performed as in Fig. 4A. (B) Parental CEM cells and derivative cells overexpressing wild-type Bcl-2 (Bcl-2 wt), phosphorylation deficient mutants of Bcl-2 (Bcl-2 S7A, Bcl-2 S87A, Bcl-2 S7A/S87A), wild-type Bcl-xL (BclxL wt) or a phosphorylation deficient mutant of Bcl-xL (Bcl-xL S62A) were stimulated with (3ng/ml). The concentrations of and, measurement of apoptosis, presentation of data, and statistical analysis were performed as described in Fig. 1A if not stated differently. Figure S3. Signaling mechanism of and impact on -induced cell death (A) Cell cycle histograms from one representative experiment from Fig. 6A are depicted of cells alive for 24 hours of incubation. Percentages indicate fraction of cells arrested in G2/M. (B) CEM cells from Fig. 5B were stimulated as indicated for 24 hours and cell cycle analysis performed as in Fig. 6A. (C) CEM cells from Fig. 6C were evaluated for the fraction of cells arrested in G2/M 24 hours after the addition of and. The concentrations of and, measurement of apoptosis, presentation of data, and statistical analysis were performed as described in Fig. 1A. p <.5, ANOVA. = not significant.. Identical signaling mechanisms in JURKAT leukemia cells as in CEM cells All functional and signaling experiments performed with CEM leukemia cells (depicted in Figs 1 7 and Figs S1 S3) were performed identically with JURKAT leukemia cells as a second independent cell line. The concentrations of and, measurement of apoptosis, Western Blot analysis, presentation of data, and statistical analysis were performed as described in corresponding figures and supplemental figures. A) See Fig. 1A. B) See Fig. S1. C) See Fig. 1B. D E) See Fig. 3A B. F,G) See Fig. S2A and B. H K) See Fig. 4A C. 2,3-DCPE was used at 1 µm, okadaic acid at,1 ng/ml. L,M) See Fig. 5A and B. N) See Fig. S3A. O,P) See Fig. 6A and B. Q,R) See Fig. S3C and Fig. 6C. S) See Fig. 6D. T) See Fig. 7.

3 36h 9,5% ,5% ,9% 11,1% ,9% ,1% ,5% 23,5% h 6,8% ,9% 5,5% 5,9% ,8% ,2% 77,2% 58,3% Figure S1

4 A Bak Bax BIM BID PUMA NOXA survivin XIAP ciap-1 ciap-2 24h 36h co d+v co d+v 24h 36h co d+v co d+v Figure S2

5 B 1 specific apoptosis (%) 5 parental Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bcl-xL Bcl-xL wt S7A S87A S7A/S87A wt S62A Bcl-2 parental Bcl-2 Bcl-2 Bcl-2 Bcl-2 wt S7A S87A S7A/S87A Bcl-xL parental Bcl-xL Bcl-xL wt S62A Figure S2

6 A 15 1 co 18,7% ,2% ,1% 84,6% B 1 co living cells in G2/M (%) 5 parental mock shp53 Figure S3

7 C 1 living cells in G2/M (%) 5 caffeine Figure S3

8 A 1 specific apoptosis (%) days

9 B 36h 13,8% ,8% 14,3% 23,6% ,8% ,7% 23,5%,5% co 48h 9,2% ,3% 8,4% 8,6% ,9% ,5% + 76,1% 48,3%

10 C Log(fa/fu) Log(d) D 12h before (combination) 2 1 Log(fa/fu) Log(d)

11 E 24h after (combination) 2 1 Log(fa/fu) Log(d) F Bak BIM BID PUMA NOXA 24h 36h co d+v co d+v survivin XIAP ciap-1 ciap-2 24h 36h co d+v co d+v

12 G 1 specific apoptosis (%) 5 parental Bcl-2 Bcl-2 Bcl-2 Bcl-2 Bcl-xL Bcl-xL wt S7A S87A S7A/S87A wt S62A Bcl-2 parental Bcl-2 Bcl-2 Bcl-2 Bcl-2 wt S7A S87A S7A/S87A Bcl-xL parental Bcl-xL Bcl-xL wt S62A

13 H Bcl-2 Bcl-xL 12h 24h co d+v co d+v p-bcl-2 Bcl-2 p-bcl-xl Bcl-xL I + + loss of mitochondrial membrane potential (%) 5 Cytochrome C release (%) h h time time 24h 36h 48h co d+v co d+v co d+v Casp-1 Casp-2 Casp-1 cl. Casp-3 cl. Casp-6 cl. Casp-7 cl. Casp-8 cl. Casp-9 cl. PARP

14 K 1 specific apoptosis (%) 5 specific apoptosis (%) 5 DCPE Bcl-2 Bcl-xL co DCPE okadaic co okadaic p-bcl-2 Bcl-2 p-bcl-xl Bcl-xL L p53 Histon H1 1h 3h 6h co d+v co d+v co d+v

15 M 1 specific apoptosis (%) 5 p53 parental mock shp parental mock shp53 N co ,4% ,7% ,1% 2 79,2% 1

16 O 1 co + cells in G2/M (%) h time P p-histon H3 Ser co 1,7% 1 5 4,2% p-histon H3 Ser p-histon H3 Ser Propidium Iodid Propidium Iodid + 79,3% 1 5 5,1% p-histon H3 Ser Propidium Iodid Propidium Iodid

17 Q 1 R living cells in G2/M (%) 5 specific apoptosis (%) 5 caffeine caffeine S 5 cells in G2/M (%) specific apoptosis (%) 5 parental mock shcyclina parental mock shcyclina cyclina parental mock shcyclina

18 T cells in G2/M (%) 5 specific apoptosis (%) 5 irradiation - + irradiation