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Additional Supporting Information may be found in the online version of this article.

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IJC_27863_sm_SuppFig1.tif279KSupporting Information Figure 1. GSI I increases CLL cell apoptosis in a dose-dependent manner. Freshly isolated CLL cells (n = 22) were exposed for 24 hr to the indicated GSI I concentrations or 0.01% DMSO. Cell viability and apoptosis were evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining, and results are presented as the percentage of viable (An V-/PI-), early apoptotic (An V+/PI-), late apoptotic (An V+/PI+), and necrotic (An V-/PI+) cells. (a) Results are the mean ± SD of 22 patients. **p < 0.01 (An V-/PI- cells at each dose of GSI I versus An V-/PI- cells in DMSO) according to Student's t test. (b) Results shown for patient 2 are representative of 22 patients.
IJC_27863_sm_SuppFig2.tif289KSupporting Information Figure 2. GSI I increases CLL cell apoptosis by potentiating an ER stress-specific caspase cascade initiated by caspase-4. (a) Effect of pharmacologic caspase inhibition on viability of CLL cells treated with GSI I. Freshly isolated CLL cells (n = 6) were pretreated for 2 hr with 50 ?M of the caspase-4 inhibitor z-LEVD-fmk, 50 ?M of the pan-caspase inhibitor z-VAD-fmk or 0.005% DMSO as control, exposed for 12 hr to 2.5 ?M GSI I, and then analyzed for Annexin V/PI (An V/PI) staining. Results are presented as the percentage of viable (An V-/PI-) cells and are the mean ± SD of 6 patients. **p < 0.01 (cells treated with each caspase inhibitor and GSI I versus cells treated with GSI I) according to Student's t test. (b) Effect of pharmacologic caspase-4 inhibition on caspase-8 and -3 processing induced by GSI I. Western blot analysis was performed in 20 ?g whole-cell lysates extracted from CLL cells treated with the caspase-4 inhibitor and GSI I as in panel a, and protein loading was assessed by reprobing the blots with an anti-GAPDH antibody (n = 6). Vertical lines inserted in the blots indicate a repositioned gel lane. Results shown for patient 2 are representative of 6 patients.
IJC_27863_sm_SuppFig3.tif348KSupporting Information Figure 3. Notch2 down-regulation by siRNA increases CLL cell apoptosis. Freshly isolated CLL cells (n = 6) were transfected with 0.5 ?M of control nontargeting (siCtrl) or Notch2 siRNA as described in “siRNA nucleofection” and then cultured for 72 hr in supernatants harvested from 48-hr cultures of OP9 cell line. (a) Western blot analysis of Notch2 was performed in 20 ?g whole-cell lysates, and protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. (b) Apoptosis was evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. Results are presented as the percentage of viable (An V-/PI-), early apoptotic (An V+/PI-), late apoptotic (An V+/PI+), and necrotic (An V-/PI+) cells. (a and b) Data of patients 2, 18 and 20 are representative of 6 patients.
IJC_27863_sm_SuppTab1.doc55KSupporting Information Table 1.
IJC_27863_sm_SuppTab2.doc42KSupporting Information Table 2.

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