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. 2018 May 29;9:1186. doi: 10.3389/fimmu.2018.01186

Figure 4.

Figure 4

Nitric oxide (NO) mediates tumor cells resistance to chemotherapeutic agent cisplatin (CDDP) through the generation of cyclic GMP (cGMP) and the inhibition of acid sphingomyelinase (A-SMase) activity. (A) Evaluation of CDDP-induced apoptosis of U373 cells cultured in the presence of CDDP (50 µg/ml, 24 h) administered alone or in the presence of DETA-NO (20 µM, 1 h before CDDP), 8Br-cGMP (3 mM, 1 h before CDDP), and DETA-NO + ODQ (1 µM, 15 min before DETA-NO). Panel on the right shows apoptosis quantification expressed as fold increase of total apoptotic cells (annexin V+/PI− and annexin V+/PI+ cells) compared to their respective untreated (UT) controls (dashed line) (n = 4). **p < 0.001 vs UT; ++p < 0.05, ++p < 0.001 vs CDDP. (B,C) U373 cells cultured in the presence of CDDP (50 µg/ml, 24 h) administered alone or together with neutralizing anti-CD95 antibody ZB4 (500 ng/ml, 1 h before CDDP). (B) Evaluation of CDDP-induced apoptosis expressed as fold increase of total apoptotic cells (annexin V+/PI− and annexin V+/PI+ cells) compared to their respective UT (dashed line) (n = 3). *p < 0.05, **p < 0.001 vs UT; +p < 0.05 vs CDDP. (C) Brightfield microscopy images representative of three independent experiments. Scale bar: 100 µm. Bottom panels represent enlarged image details marked by the white arrows. (D) A-SMase activity on cell lysates measured as sphingomyelin hydrolysis to phosphorylcholine at pH 5.5. Cells (n = 3) were treated for the indicated time points (30, 60, and 120 min) with CDDP (50 μg/ml). Enzyme activity is expressed as fold increase compared to UT (dashed line). *p < 0.05, **p < 0.001 vs UT. (E) A-SMase activity on cell lysates derived from cells cultured in the presence of CDDP (50 μg/ml, 30 min) administered alone or together with DETA-NO (20 µM, 1 h before CDDP), 8Br-cGMP (3 mM, 1 h before CDDP), and DETA-NO + ODQ (1 µM, 15 min before DETA-NO) (n = 3). Enzyme activity is expressed as fold increase compared to UT controls (dashed line). **p < 0.001 vs UT; ++p < 0.001 vs CDDP. (F) A-SMase translocation was evaluated, at the indicated time points after CDDP (50 μg/ml) administered alone or together with DETA-NO (20 µM, 1 h before CDDP), 8Br-cGMP (3 mM, 1 h before CDDP), and DETA-NO + ODQ (1 µM, 15 min before DETA-NO), by assessment of biotinylated plasma membrane A-SMase, using the A-SMase antibody and assessing cytosolic GAPDH expression in parallel as internal control. The images are representative of three independent experiments.