Figure 5

Hypertonicity and SMAC mimetics cooperate in autocrine TNF release. (a) L929 cells were cultured with or without addition of 75 mM NaCl and treated with the indicated concentrations of BV6 and LCL161 for 18 h. Supernatants were collected and secretion of murine TNF was quantified using ELISA. (b) REH cells were cultured with or without addition of 50 mM NaCl and treated with the indicated concentrations of BV6, LCL161 and birinapant (Biri) for 18 h. Secretion of human TNF in the supernatant was quantified using ELISA. Data shown for (a) and (b) are mean values±S.E.M. from three independent experiments. (c) REH cells were challenged with 1.25 μM birinapant in the presence and absence of 50 mM NaCl for the indicated periods of time. After washing and lysis, western blot analyses were performed with antibodies specific for the indicated proteins. Detection of tubulin served as a loading control. (d) REH cells were challenged with 50 mM NaCl or 1.25 μM birinapant (Biri) for 6 h and subsequently analyzed by western Blotting for the presence of the indicated proteins. (e) L929 cells were challenged with the indicated concentrations of NaCl for 18 h. Secretion of murine TNF in the supernatant was quantified using ELISA. Data shown are representative of two experiments performed. (f) REH cells were challenged with 50 mM NaCl for the indicated periods of time or left untreated. Tnf mRNA induction was analyzed by qPCR. Data points of three independent experiments together with mean±S.E.M. are shown. Biri, birinapant