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

Figure 6.

Figure 6

Nitric oxide (NO)/cyclic GMP (cGMP) induces degradation of syntaxin 4 (synt4) by the proteasome via protein kinase G-dependent phosphorylation of Ser-78 which explains the chemoresistance of tumor cells. (A) Synt4 expression in U373 cells treated with 8Br-cGMP (3 mM, 1 h) in the presence or in the absence of MG132 (10 µM), assessed by western blotting. The images are representative of three independent experiments. Panel on the right shows the densitometry analysis of synt4 expression. Data are expressed as the fold change over their respective controls (UT or MG132 alone) (dashed line). **p < 0.001 vs UT. (B) Immunoprecipitation of ubiquitinated synt4. Cells were treated with 8Br-cGMP (3 mM, 1 h) in the presence of MG132 (10 µM, 2 h before 8Br-cGMP administration), lysed, and incubated with Agarose-TUBEs to immunoprecipitate ubiquitinated protein. Synt4 expression was detected by western blotting. The images are representative of three independent experiments. (C) Synt4 expression in U373 cells treated with 8Br-cGMP (3 mM, 1 h) in the presence or in the absence of KT5823 (1 µM, 15 min before 8Br-cGMP administration), assessed by western blotting. The images are representative of three independent experiments. Panel on the right shows the densitometry analysis of synt4 expression. Data are expressed as the fold change over their respective controls (UT or KT5823 alone) (dashed line). **p < 0.05 vs UT. (D) Synt4 expression in cells transfected with synt4 wild-type and synt4 mutant proteins. GAPDH was used as the internal standard. The images are representative of three independent experiments. (E) Acid sphingomyelinase activity on cell lysates derived from cells transfected with the mutant protein S78A and treated with chemotherapeutic agent cisplatin (CDDP) (50 µg/ml, 30 min) alone or in the presence 8Br-cGMP (3 mM, 1 h before CDDP administration) (n = 3). Enzyme activity is expressed as fold increase compared to UT controls (dashed line). (F) Evaluation of CDDP-induced apoptosis of U373 cells transfected with the mutant protein S78A and treated with CDDP (50 µg/ml, 24 h) alone or in the presence 8Br-cGMP (3 mM, 1 h before CDDP administration). 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 UT controls (n = 4).