FIGURE 5: TL-532 induces both TLR3-dependent inflammatory/immunogenic cell death by apoptosis in NCI-H292 wt cancer cells and mRANTES/mIP10 secretion in RAW264.7 wt. (A-C) NCI-H292 wt cells and/or its TLR3 KO counterpart were treated with TLR3-ligands at indicated doses for 24 hours. TLR3-agonist activity was determined: by the fold apoptosis compared to untreated cell lines using AnnexinV-Glo assay (A), Cell viability by MTS assay (B), and cytokine secretion by hIL6-ELISA (C). (D) The apoptotic nature of cell death in NCI-H292 wt cells was determined by adding or not the pan-caspase inhibitor Z-VAD-FMK (20 µM for 2 hours) prior to TL-532 treatment at 100 µg/mL for 6 hours. Percentage of AnnexinV+/Pi+ cells was analyzed by flow cytometry. (E-F) In vitro immunogenic cell death was determined in NCI-H292 cells by quantifying the following biomarkers in cell culture supernatant: ATP at 4 hours post-treatment using ATP-Glo assay (E) and HMGB1 by ELISA at 24h hours post treatment, expressed as the percentage of released HMGB1 compared to the total amount of intracellular HMGB1 (F). (G-H) RAW264.7 wt cells were pretreated with the pan-cathepsin inhibitor Z-FA-FMK (50 μM for 48hr) before treatment with 500 µg/mL of Poly(I:C) HMW and Poly(A:U) HMW and TL-532, and with 1 µg/mL of LPS, for 24 hours. The concentration of mRANTES (G) and mIP10 (H) was measured by ELISA. Data are the mean of three independent assays (A-H). Results are expressed as mean ± 95% confidence interval (A, B, C, E, G, H). Results are expressed as mean ± SD (D, F). Unpaired Student's t-test: * = p<0.05; ** = p<0.01; *** = p<0.001; ns: not significant.