Fig 2: Knockdown of c-FLIP induces apoptosis and its introduction averts apoptotic cell death in the starvation-resistant RCC cell line SW839(A) Apoptotic signals of CellEvent™ Caspase-3/7 Green Detection Reagent (apoptotic cells, green) and Hoechet33342 (nuclei of total cells, blue) in the starvation-resistant RCC cell line SW839 under 1-day and 2-day treatments with the indicated siRNA reagents (siCont, siHIF2 and siFLIP). (B) Photographic representation of SW839 cells, in which ß-gal or c-FLIP was introduced lentivirally, after 2-day treatment with siHIF2. Note that knockdown of c-FLIP induced similar or greater apoptosis, and exogeneous introduction of c-FLIP completely averted apoptosis with siHIF2 in SW839 cells. These are photographic representations of data shown in Figure 4D and 4E.

Fig 3: TAS4464 exerts strong antitumor activity via activation of both the intrinsic and extrinsic apoptotic pathways accompanied by an increase in the NOXA level and a decrease in the c-FLIP level in a human AML xenograft model.A TAS4464 (100 mg kg-1) was administered intravenously. Tumors were harvested at the indicated times after administration of TAS4464 and prepared for immunoblotting. B TAS4464 was administered intravenously twice weekly at 100 mg kg-1 per day. Cytarabine was administered intravenously twice a week for 3 weeks at 100 mg kg-1 per day. Data are presented as the mean ± SD values. *P < 0.05 in the treated group compared with the control group (Dunnett’s test).

Fig 4: The cFLIPL/calmodulin interaction is mediated by the DED1 domain.Calmodulin conjugated on sepharose resin was used as bait in all pull-down assays and purified recombinant cFLIPL or its DED domains were used as prey (10 g each). Pull-down lanes consisted of 50 µL resin and supernatant lanes of 500 µL buffer, rendering a dilution factor of 10 for non-interacting prey proteins in the SDS/Coomassie gel. (A) Full-length cFLIPL pulls down on calmodulin-conjugated resin (lane 2), with DED1 strongly interacting with calmodulin (lane 3) while DED2 exhibits almost no interaction (lane 4). GST alone, used as a control, displayed no binding to calmodulin (lane 1). (B) The pull-down was repeated for DED1 and full-length cFLIPL in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of Ca2+. Neither cFLIP nor its DED1 interact with calmodulin in the absence of Ca2+ (lanes 2 and 4). Only pull-down lanes are shown. (C) To further confirm the specificity of the DED1/calmodulin interaction, a competition assay was carried out in the pull-down format, illustrated conceptually in this schematic. Recombinant GST-DED1 is incubated with calmodulin-conjugated resin in the presence of increasing amounts of free calmodulin, which is expected to compete with the resin for binding to DED1. (D) Results of assay described in C. Free calmodulin displaces DED1 from the resin in a dose-dependent manner.

Fig 5: Expression of c-FLIP following cflar transfection and subsequent LPS and CHX induction. (A) Expression of c-FLIP in the transfected cflar human umbilical vein endothelial cells was higher, compared with that in cells transfected with PEGFP-N1 alone, confirmed using western blot analysis. (B) Following transfection only, the expression of c-FLIP decreased at 36+9 h (#P<0.05, compared with 36+0 h), and then gradually increased with increasing transfection duration, particularly at 36+24 h (*P<0.05, compared with the 36+6, 36+9 and 36+12 h groups, respectively). (C) Expression of c-FLIP increased, and then decreased following treatment of untransfected cells with LPS+CHX, but without statistical significance. (D) Expression of c-FLIP increased significantly at 9 h post-transfection in the LPS+CHX group (#P<0.05, compared with 36+0 h). (E) Expression of c-FLIP in cells 9 h post-transfection with LPS or LPS+CHX. The LPS group showed higher protein expression of c-FLIP, compared with the control group (P=0.0421). The LPS+CHX group showed higher protein expression of c-FLIP, compared with the control and LPS groups (P=0.0286 and P=0.0421, respectively). (F) Representative images of western blot analysis of protein expression of c-FLIP and ß-actin in the three groups. c-FLIP, cellular FLICE-inhibitory protein; LPS, lipopolysaccharide; CHX, cycloheximide; cflar, caspase-8 and Fas-associated death domain-like apoptosis regulator.