Fig 2: ATP inhibits soluble TNF release. A) RAW 264.7 macrophages exposed either LPS alone (1 µg/ml, 2 h) or 2-hit injury model with LPS (1 µg/ml, 1 h) followed by ATP (3 mM, 1 h). LPS induced soluble TNF secretion into cell- and MV-depleted supernatants, which was almost entirely abolished by ATP treatment (left, n = 9–10). ATP reduced both surface [middle, represented as mean fluorescence intensity (MFI), n = 5–6] and total cellular TNF expression (right, n = 5–6). B) Confocal images clearly showed disappearance, rather than accumulation, of cellular TNF from the RAW macrophages upon ATP exposure (n = 3). C) To confirm these results in primary cells, we exposed mouse BMDMs to LPS (1 µg/ml, 1 h) followed by ATP (3 mM, 15 min). Similarly, ATP inhibited soluble TNF secretion (left, n = 6) and reduced total cellular TNF expression (middle and right, n = 6). D) Confocal images of BMDMs illustrating the reduction of cellular TNF expression following ATP treatment (n = 3). Cells were also checked for TNF expression under nonstimulated conditions (i.e., in PBS or medium) and, as expected, TNF was not detected in this sterile or noninflamed environment (Supplemental Fig. S1G). Parametric or nonparametric data are displayed as means ± sd or box-whisker plots showing the median, IQR, and minimum or maximum values, respectively. Scale bars, 10 µm. **P < 0.01, ***P < 0.001.

Fig 3: Hyperosmotic stress enhances SMAC mimetic-induced, TNF-mediated apoptosis in ALL cells. (a and b) REH cells were grown in medium supplemented with 50 mM NaCl and challenged with the indicated concentrations of (a) LCL161 and (b) birinapant (Biri) in the presence and absence of etanercept (400 µg/ml). Shown are data points and mean±S.E.M. from three independent experiments. (c) REH cells were left untreated or challenged with 2.5 µM LCL161 or 2.5 µM birinapant with or without addition of 50 mM NaCl. After incubation for 16 h, cells were analyzed by flow cytometry for positivity of 7-AAD and annexin-V. Data shown are representative of two experiments performed. (dand e) REH cells were challenged with 0.3 µM BV6, LCL161 and birinapant with or without addition of 50 mM NaCl for 18 h. Caspase-8 and caspase-3/-7 activity was assessed using the fluorogenic substrates (IETD)2-R110 and (DEVD)2-R110, respectively. Data points from three independent experiments are shown. A line connects caspase activation under isotonic conditions with the corresponding value under hypertonic conditions (50 mM NaCl) for each experiment. (f) REH cells were treated with the indicated concentrations of BV6, LCL161 and birinapant with or without addition of 50 mM NaCl and 50 µM zVAD-fmk. Shown are data points and mean±S.E.M. from three independent experiments. Biri, birinapant

Fig 4: Hypertonicity-induced TNF release overcomes resistance to SM monotherapy. (a) TF-1 cells were challenged in the presence and absence of 50 mM NaCl with the indicated concentrations of birinapant. Shown are data points and mean±S.E.M. from three independent experiments. (b) Co-cultures of TF-1 and REH cells under iso- and hypertonic (+50 mM NaCl) conditions were challenged with the indicated concentrations of birinapant in the presence and absence of etanercept (1 mg/ml). Shown are data points and mean±S.E.M. from three independent experiments. (c) TF-1 and REH cells were challenged with 10 µM birinapant in the presence of 50 mM NaCl for 18 h or left untreated. Supernatant of REH cells was harvested and transferred onto TF-1 cells. After 7 h, viability of TF-1 cells was measured using MTT. Shown are data points and mean±S.E.M. from four independent experiments. Biri, birinapant; SN, supernatant

Fig 5: PC attenuation of angiotensin II-induced inflammation and oxidative stress in H9C2 cells. H9C2 cells were treated with PC (50 and 100 µg/ml) for 2 h before coincubation with angiotensin II (1 µM) for 48 h. mRNA expression of NFKB1 (a), TNF (b), SOD1 (c), GPX1 (d), and FOXO3 (e) were measured by qRT-PCR. Experimental data were shown as mean ± standard deviation (SD). n = 3. *P < 0.05 and **P < 0.01.