Journal: Molecular cancer therapeutics

Article Title: The Functionalized Human Serine Protease Granzyme B/VEGF 121 Targets Tumor Vasculature and Ablates Tumor Growth

doi: 10.1158/1535-7163.MCT-13-0165

Figure Lengend Snippet: (A–B) GrB/VEGF121 activates caspase-3 (4A) and -9 (4B) in whole cell lysates. Chromogenic substrates for caspase-3 and caspase-9 were incubated in PAE/KDR whole cell lysates with or without GrB or GrB/VEGF121. Activation of the caspases was indicated by an increase in absorbance of the cleaved substrate. (C) Pre-incubation of PAE/VEGFR-2 cells with z-VAD-fmk (20 or 100 μM) for 1 h prior to GrB/VEGF121 treatment for 2 h significantly blocked caspase-3 activation. (D–E) GrB/VEGF121-mediated cytotoxicity on PAE/VEGFR-2 and PAE/VEGFR-1 cells over 72 h is unchanged despite co-incubation with various concentrations of z-VAD-fmk. (F) Granzyme B internalization results in caspase-3 cleavage in vitro. PAE/VEGFR-2 cells were pre-treated with up to 100 μM z-VAD-fmk prior to addition of 20 nM GrB/VEGF121 for one hour. GrB/VEGF121-mediated cleavage of caspase-3 cleavage occurred in the presence of up to 100 μM z-VAD-fmk. (G–H) Caspase-independent PARP cleavage. PAE/VEGFR-2 and PAE/VEGFR-1 cells were treated with up to 48 with 20 nM GrB/VEGF121 in the presence or absence of 20 μM z-VAD-fmk. PARP cleavage was observed in PAE/VEGFR-2, but not PAE/VEGFR-1, cells.

Article Snippet: Substrate specificity was assessed incubation with the pan-caspase inhibitor z-VAD-FMK (Enzo Life Sciences).

Techniques: Incubation, Activation Assay, In Vitro

Journal: Oncotarget

Article Title: HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression

doi: 10.18632/oncotarget.17157

Figure Lengend Snippet: (a) siRNA transfected Panc-1 cells were treated with various doses of TRAIL for 6 h. Protein lysates from whole cells were assayed using immunoblotting. α-tubulin was used as a loading control. (b) siRNA transfected Panc-1 cells were cultured with TRAIL (100 ng/mL) in the presence of the indicated caspase inhibitors (20 μM). The cells were examined using flow cytometric analysis. The number represents the percentages of each subset. zVAD, pan-caspase inhibitor (z-VAD-FMK); C8i, caspase-8 inhibitor (z-IETD-FMK); C9i, caspase-9 inhibitor (z-LEHD-FMK). Similar results were obtained from two independent experiments. (c) The results of Annexin V + cells (%) are presented as means ± SD from triplicate experiments. ** p < 0.01. (d) siRNA transfected Panc-1 cells were treated with the indicated concentration of TRAIL for 6 h. The protein expression levels of c-FLIP were determined using immunoblotting. α-tubulin was used as a loading control. (e) Similarly, the lysates were used for immunoblotting to examine the expression of the indicated proteins. β-actin was used as a loading control.

Article Snippet: The following caspase inhibitors were added to the in vitro proliferation assay 2 h before adding TRAIL at a dose of 20 μM: pan-caspase inhibitor z-VAD-FMK (Enzo Life Sciences), caspase-8 inhibitor z-IETD-FMK (R&D Systems), and caspase-9 inhibitor z-LEHD-FMK (R&D Systems).

Techniques: Transfection, Western Blot, Cell Culture, Concentration Assay, Expressing