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

Journal: Animal Cells and Systems

Article Title: The effect of caspase-3 inhibition on interdigital tissue regression in explant cultures of developing mouse limbs

doi: 10.1080/19768354.2012.678386

Figure Lengend Snippet: Figure 2. Caspase-3 correlation with apoptosis and proliferation in the mouse front limbs in vivo (AL) and quantification of active caspase-3 positive cells (MO). Collocalization of caspase-3 immunohistochemistry (IHC) (A, D, G, and J), TUNEL assay (B, E, H, and K), and PCNA immunohistochemistry (C, F, I, and L) in serial sections at E12.5 (AC), at E13.5 (DF), at 14.5 (GI), and at 15.5 (JL). Positive cells are brown (black arrows), negative blue (white arrows), magnification 200. Flow cytometry of interdigital (M) and digital (N) cells at 13.5 and percentage comparisons after multiple measurements (O).

Article Snippet: Penetration of the inhibitor in the culture was confirmed using a biotinylated pan caspase inhibitor (R&D System, FMK 011) (data not shown).

Techniques: In Vivo, Immunohistochemistry, TUNEL Assay, Flow Cytometry

Journal: Animal Cells and Systems

Article Title: The effect of caspase-3 inhibition on interdigital tissue regression in explant cultures of developing mouse limbs

doi: 10.1080/19768354.2012.678386

Figure Lengend Snippet: Figure 3. Pharmacological inhibition of caspase-3 in mouse limb explant cultures. (AC) control group, (DF) inhibited group, magnification 200. (A and D) Caspase-3 activation in serial sections after 24 hours of cultivation based on IHC shows no active caspase-3 in the inhibited samples. (B and E) TUNEL assay in serial sections after 24 hours of cultivation shows dramatic reduction of apoptosis in the inhibited samples. (C and F) Comparable growth of the explants cultures checked using PCNA immunohistochemistry (IHC). (G) Macroscopic view on the cultivation starting point stage limb. (H) Control limb after 48 hours of cultivation with loss of the interdigit tissue. (I) Caspase-3-inhibited explant showing interdigital webbing. (J) Caspase-3 was negative after 72 h of cultivation but (K) TUNEL positive cells reappeared and (L) digit separation continued.

Article Snippet: Penetration of the inhibitor in the culture was confirmed using a biotinylated pan caspase inhibitor (R&D System, FMK 011) (data not shown).

Techniques: Inhibition, Control, Activation Assay, TUNEL Assay, Immunohistochemistry