Fig 2: Wild type mouse-derived immortalized fibroblasts, as well as TRAILR1- and TRAILR2-positive transfectants lack sensitivity to TRAIL-induced cell death. A. Mouse fibroblasts are nonresponsive to the cytotoxic effects of TRAIL. Cells were treated with FLAG-TRAIL (400 ng/ml) in the absence or presence of the crosslinking anti-FLAG antibody M2 (2 µg/ml; 1 h 37°C) and/or 0.5 µg/ml cycloheximide, as indicated. Cell viability was determined by crystal violet staining the next day. One representative experiment (n = 3, shown are the mean values ± standard deviation (SD)) out of three is shown. B and C. Immortalized mouse fibroblasts were stably transfected with wild-type human TRAILR1 (B) or TRAILR2 (C) expression plasmids. Cell viability was determined by crystal violet staining following 16 hour incubation with cross-linked FLAG-TRAIL in the absence (open symbols) or presence (closed symbols) of cycloheximide (0.5 µg/ml). One representative experiment (n = 3, shown are the mean values ± SD) out of three is shown. Caspase-8 (dashed bars) and caspase-3 (black bars) enzymatic activity following triggering of indicated receptors with FLAG-TRAIL (previously cross-linked with anti-FLAG M2 antibody, 1 h 37°C) was quantified using specific fluorogenic substrates (Ac-IEPD-AMC and Ac-DMQD-AMC, respectively). The insets show the expression pattern of the human TRAIL receptors as analyzed by flow cytometry. One representative experiment (out of three) is shown.

Fig 3: Non-responders can be sensitized to IZI1551-induced cell death by Mcl-1 antagonism.A Quantification of cell death kinetics, calculated as a percentage of PI-positive cell areas. Cells were co-treated with IZI1551 (1 nM) and marizomib (80 nM) simultaneously or pre-treated with marizomib for 24 h (MRZ −24 h) before the addition of IZI1551. Representative results from one out of 3 independent experiments are shown. Error bars represent the SD of 3 technical replicates. B Marizomib inhibited proteasome activities both in responder and non-responder cell lines. Cells were treated with 40 nM (low) or 80 nM of marizomib or bortezomib for 4 h and CT-L activities of the proteasomes were measured from total cell lysates by cleavage of Suc-LLVY-AMC. Bars indicate % of activity related to untreated controls. Data are shown as mean ± range of n = 2 measurements. C Surface expression of death receptors DR4 and DR5 in responsive and resistant cell lines. Cells were treated with marizomib (80 nM) and surface amounts were determined by flow cytometric measurements. Data show mean plus range. Similar results were obtained in independent repeat experiments. D Procaspase-8; FADD; FLIP and BID proteins are detected at different expression levels in responsive and resistant cells. Cells were treated with marizomib and the indicated proteins were detected in whole-cell lysates. Vinculin served as a loading control. Similar results were obtained in independent repeat experiments. E Anti-apoptotic Bcl-2 family proteins were detected in different cell lines. Cells were treated with marizomib (80 nM) and the indicated proteins were detected in whole-cell lysates. Vinculin or α-Tubulin served as loading controls. Similar results were obtained in independent repeat experiments. F Annexin V/PI-based flow cytometry of cells treated with 10 μM of S63845; 1 nM IZI1551 plus 80 nM MRZ or the combination thereof for 24 h. Data represent mean ± SEM of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001; one-way ANOVA followed by Tukey post hoc test.

Fig 4: Cancer cell cytotoxicity of TRAIL-ATNCs via TRAIL receptors. (A) Cell viability following treatment with TRAIL, TRAIL-ATNC, or TRAIL-ATNCIL4rP. MDA-MB-231 cells were incubated with proteins for 24 h and then measured by EZ-count assay. Data represent means ± SEM. (B) The apoptosis inducing ability of TRAIL or TRAIL-ATNC was analyzed with Annexin V and propidium iodide (PI) double staining using flow cytometry. The percentage of annexin V/PI double-negative (viable) cells was plotted. (C) Apoptosis inducing ability of TRAIL or TRAIL-ATNC was blocked by anti-DR4/DR5 antibodies. MDA-MB-231 cells were pre-incubated with DR4/DR5 blocking antibody or control IgG for 1 h and were incubated with TRAIL or TRAIL-ATNC for 3 h. Cell viability was measured with Annexin V and PI double staining using flow cytometry.

Fig 5: Oxygen and nutrient deprivation result in accumulation of TRAILR2.a, b Cell lysates of 2D-cultivated cells or cells grown as spheroids (day 11) were analyzed by western blotting. Blots are representative of three independent experiments. c Paraffin-embedded spheroid slices (day 11) were immunohistochemically stained for Hif-1α and counterstained with hematoxylin. Cells were color coded as positive (yellow) or negative (blue). Pictures are representative of three independent experiments. Scale bars = 200 μm. d Percentages of cells positive or negative for Hif-1α expression are shown as mean values ± SD of 4–16 spheroids from 3 independent experiments. Asterisks indicate statistical significance (***p ≤ 0.001; unpaired t-test). e Lysates from cells cultivated at 1% O2 were analyzed by western blotting. Blots shown are representative of three independently performed experiments. f TRAILR2 surface expression measured by flow cytometry. Medians of the cell populations were used to calculate surface expression relative to cells cultivated at control conditions. Data are mean values ± SD of at least three independent experiments. g Lysates from cells cultivated at starvation conditions were analyzed by western blotting. Blots shown are representative of three independently performed experiments. h TRAILR2 surface expression measured by flow cytometry. Medians of the cell populations were used to calculate surface expression relative to cells cultivated at control conditions. Data are mean values ± SD of at least three independent experiments.