Journal: Oncology Reports

Article Title: Expression level of Bcl-XL critically affects sensitivity of hepatocellular carcinoma cells to LIGHT-enhanced and interferon-γ-induced apoptosis

doi: 10.3892/or.17.5.1067

Figure Lengend Snippet: Figure 3. Expression levels of caspase-3, caspase-8, caspase-9, Bcl-XL, Bak and Bid in Hep3B cells treated with IFN-Á/LIGHT. (A) Hep3B cells were treated with 100 ng/ml IFN-Á and various concentrations of LIGHT for 24 h or 72 h as indicated. Cell lysates containing 30 μg of protein were subjected to 12% Tris-glycine gel electrophoresis followed by Western blotting with rabbit polyclonal antibodies against caspase-3, caspase-8, caspase-9, Bcl- XL, Bak, Bid, respectively. Cells treated with 100 ng/ml IFN-Á or 100 ng/ml LIGHT alone and untreated cells were used as the controls. ß-actin was used as an internal control for total protein loading. (B) Hep3B cells (2x105) treated with 100 ng/ml sLIGHT and 100 ng/ml of IFN-Á were also incubated with 100 μM of caspase-3 inhibitor Z-DEVD-fmk, caspase-9 inhibitor Z- IEHD-fmk, caspase-8 inhibitor Z-IEHD-fmk, or normal saline for 96 h. Cell viability was then measured. The viability of untreated (without any cytokine and inhibitor) cells was set at 100%, and the relative viability of other samples was calculated accordingly. The experiments were performed in triplicate, and in at least two separated experiments. The bar indicates the standard error. **p<0.01, versus group treated with normal saline.

Article Snippet: A recombinant LIGHT soluble protein that only contained the extracellular region of human LIGHT and caspase inhibitors Z-DEVD-fmk, Z-IETD-fmk and Z-LEHD-fmk was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot, Control, Incubation, Saline

Journal: Oncotarget

Article Title: YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network

doi:

Figure Lengend Snippet: Ex vivo cultures of primary human breast tumors ( n = 19) were cultured 48 h with 50 nM YM155 or not treated (untreated). Samples were then analyzed for cancer cell morphology by Hematoxylin-Eosin-Saffron staining (upper panel) and active caspase-3 positivity by immunohistochemistry lower panel), as shown in A. for a YM155-responsive (left panel) or a resistant tumor (right panel). B. Data including 19 tumors are represented as % of carcinomatous cells positive in each specimen, in both untreated and YM155-treated conditions.

Article Snippet: YM155, Necrostatin-1 and BAY11-7085 were purchased from Selleck Chemicals (Houston, USA) and the pan-caspase inhibitor Q-VD-OPh from R&DSystems (Abingdon, UK).

Techniques: Ex Vivo, Cell Culture, Staining, Immunohistochemistry

Journal: Oncotarget

Article Title: YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network

doi:

Figure Lengend Snippet: A. MDA-MB321 or Cal51 cells were treated by YM115 at 40 nM and 70 nM respectively for 48 h and cleavage of caspase-3 and Bax proteins were evaluated by immunoblot analysis. The arrow on Bax immunoblot indicates a cleaved form of Bax with 18KDa MW. B. MDA-MB321, MCF-7 and Cal51 cells were first incubated with 10 μM of the pan-caspase inhibitor QVD-Oph (Q) and/or with 1 μM of the RIP1 inhibitor Necrostatin-1 (N) for 3 h then treated with respectively 50, 40 or 70 nM of YM155 for 48 h in presence of the above inhibitors (YM155) or not (untreated). Cell death assays were then performed and % of dead cells determined in each indicated condition. No significant inhibition of cell death was observed ( n = 3). C. A similar pretreatment with the autophagy inhibitors Chloroquine (25 μM) or 3-MA was applied to MDA-MB231, Cal51 or MCF-7 cells, followed by a 48 h-treatment by YM155 at above concentrations (YM155) or not (Un), before cell death evaluation. D. Similar culture conditions were applied before performing immunoblot analysis for LC3 evaluation. When indicated, Chloroquine pretreatment (25 μM) was realized 3 h before adding YM155. E. LC3 was evaluated by IHC in YM155-treated (+) at 40 nM for 48 h versus untreated (−) Cal51 cells.

Article Snippet: YM155, Necrostatin-1 and BAY11-7085 were purchased from Selleck Chemicals (Houston, USA) and the pan-caspase inhibitor Q-VD-OPh from R&DSystems (Abingdon, UK).

Techniques: Western Blot, Incubation, Inhibition