Fig. 4

ViscumTT induces cellular stress responses. a TC-71 and MHH-ES-1 cells were treated with increasing concentrations of viscumTT (ML-I 1–40 ng/mL + OA 10–60 μg/mL), viscum (ML-I 1–40 ng/mL) or TT (10–60 μg/mL) for 24 h. Activation of stress-mediated MAPK signalling (p-MAPK8, p-MAPK14), cellular stress/unfolded protein response (EIF2AK3, HSPA5) and autophagy (LC3BI/II) were assessed using western blotting. β-actin was used as loading control, and images shown are representative for three independent experiments. Mistletoe lectin (ML) and oleanolic acid (OA) concentrations were used as a measure of viscum and TT active agent extract concentration, respectively. b TC-71 cells were treated with viscumTT, viscum or TT (~ IC50 concentration) for 24 h in the presence of DMSO (inhibitor solvent control), 10 μM SB203580 or 5 μM SP600125 to inhibit MAPK14 or MAPK8 activation, respectively. Inhibition of MAPK activation was not detected within the used inhibitor concentrations in whole-cell extracts using western blotting. β-actin was used as loading control, and images are representative for results from three independent experiments. c TC-71 cells were treated with viscumTT, viscum or TT (~ IC50 concentration) for 24 h in the absence or presence of the TLR4 inhibitor, LPS-RS (0.1 μg/mL), or the antioxidant, N-acetylcysteine (NAC, 5 mM). Apoptosis was flow cytometrically assessed following annexin V/propidium iodide staining. Bars show the percentage of apoptosis inhibition is shown in bars (±SEM) from three independent experiments