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Fig. 5 | Breast Cancer Research

Fig. 5

From: Combined the SMAC mimetic and BCL2 inhibitor sensitizes neoadjuvant chemotherapy by targeting necrosome complexes in tyrosine aminoacyl-tRNA synthase-positive breast cancer

Fig. 5

Combination treatment with SM/z-VAD and BCL2 inhibitor promotes tumor death via phosphorylation of necrosome complex in YARS-positive breast cancer cells. a The cytotoxicity effect of SM (T47D, 0.5 μM; MDA-MB-231, 2 μM) in the absence or presence of 10 μM z-VAD.fmk on 3D spheroid models. Cell death was assessed using 3D CellTiter-Glo Luminescent Cell Viability (top). YARS knockdown cells were treated with SM for 24 h in the absence or presence of 10 μM z-VAD.fmk, and cell death was determined by FSC/SSC analysis and flow cytometry (bottom). b Cells were treated with SM for 24 h in the absence or presence of z-VAD.fmk. Cell death was determined by PI staining followed by flow cytometry (right). Representative graphs of cell death determined using PI staining (left). YARS knockdown cells were treated with SM for 24 h in the absence or presence of 10 μM z-VAD.fmk., and cell death was determined by PI staining followed by flow cytometry (lower right). Representative graphs of cell death determined using PI staining (left). c The mode of cell death after combination treatment with SM/z-VAD.fmk was determined by Annexin V/PI staining and flow cytometry (left). Representative graphs show the distribution of Annexin-V- and/or PI-positive and PI-negative cells (right). d Cells were pre-stimulated with DMSO (control), z-VAD.fmk (10 μM) and/or Nec-1 (50 μM), GSK’872 (5 μM), and NSA (1 μM) in several combinations for 2 h followed by stimulation with SM for a further 24 h. Cell death was determined by PI staining and flow cytometry (left). The representative graph shows the distribution of PI-positive cells for each drug combination (right). In ad, the mean ± SD of at least three independent experiments performed in triplicate are shown; *P < 0.05; **P < 0.01; ***P < 0.001. e 3D spheroids were treated for 24 h with SM (T47D, 0.5 μM; MDA-MB-231, 2 μM) in the presence of 10 μM z-VAD.fmk and/or 50 μM Nec-1, 5 μM GSK’872, or 1 μM NSA. Cell death was assessed by 3D CellTiter-Glo Luminescent Cell Viability. f The protein levels of RIPK, p-RIPK, RIPK3, p-RIPK3, MLKL, and p-MLKL were assessed by western blotting in the presence of SM/z-VAD and/or H2O2. GAPDH served as a loading control. g Drug combination experiments assessing the effect of the treatment of 3D spheroids with the indicated drugs, ABT-263 (T47D, 1 μM; MDA-MB-231, 0.5 μM) and ABT-199 (1 μM), combined with SM/z-VAD.fmk treatment on YARS-induced cell death. Cell death was assessed using 3D CellTiter-Glo Luminescent Cell Viability. h For determining the combined effect of ABT-263 and SM/z-VAD.fmk on the YARS-induced necroptosis 3D spheroid models, the models were treated with ABT263 (T47D, 1 μM; MDA-MB-231, 0.5 μM) and/or DTX (T47D, 5 nM; MDA-MB-231, 10 nM), ADR (500 nM), or CPM (T47D, 0.5 mM; MDA-MB-231, 1 mM). 3D cell viability was analyzed using a CellTiter-Glo Luminescent Cell Viability Assay Kit (left), and T47D-derived spheroid images were assessed using phase-contrast microscopy (right). Data (e, g, h) shown are the mean ± SEM with n = 4; *P < 0.05; **P < 0.01; ***P < 0.001

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