ATPγS induces NADPH oxidase-dependent COX-2 expression in A549 cells.

<p>Cells were pretreated with Edaravone, DPI, or APO for 1 h, and then incubated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE₂ release. Cells were labeled with DCF-DA (10 µM), and then incubated with ATPγS (100 µM) for (C) the indicated time intervals or (D) pretreated with Edaravone (10 µM), DPI (10 µM), or APO (100 µM) for 1 h, and then stimulated with ATPγS (100 µM) for 1 h. The fluorescence intensity (relative DCF fluorescence) was measured and NADPH oxidase activity was determined. Cells were treated with (E) ATPγS for the indicated time intervals or (F) pretreated with Edaravone (10 µM), DPI (10 µM), or APO (100 µM) for 1 h, and then stimulated with ATPγS (100 µM) for 1 h. After incubation, CellROX™ Deep Red Reagent was added at a final concentration of 5 µM to the cells, and then incubated for 30 min at 37°C. Subsequently, medium was removed and the cells were washed thrice with PBS. The resulting fluorescence was measured using a fluorescence microscope. (G) Cells were incubated with ATPγS (100 µM) for the indicated time intervals. The membrane and cytosolic fractions were prepared and analyzed by western blot using an anti-p47<i>^phox</i> antibody. (H, I) Cells were transfected with siRNA of scrambled or p47<i>^phox</i>, and then incubated with ATPγS for 6 h. The levels of p47<i>^phox</i> and COX-2 expression were analyzed by western blot. The media was collected and analyzed for PGE₂ release. Data are expressed as mean±S.E.M. of three independent experiments. *<i>p</i><0.5; ^#<i>p</i><0.01, as compared with the cells exposed to ATPγS alone (A, B, D), the cells exposed to vehicle alone (C), or the cells transfected with scrambled siRNA and exposed to ATPγS alone (H, I).</p

ATPγS induces P2 receptor-dependent COX-2 expression, PKC translocation, and ROS generation in A549 cells.

<p>Cells were pretreated with PPADS or suramin for 1 h, and then treated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE₂ release. (C) Cells were labeled with DCF-DA (10 µM), pretreated with PPADS or suramin for 1 h, and then incubated with ATPγS for 1 h. The fluorescence intensity (relative DCF fluorescence) was measured (gray bar). In addition, NADPH oxidase activity was determined (white bar). (D) Cells were pretreated with PPADS or suramin for 1 h, and then incubated with ATPγS for 1 h. After incubation, ROS generation was determined by using CellROX™ Deep Red Reagent as described in Fig. 2E. (E) Cells were pretreated with PPADS (10 µM) or suramin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. The membrane and cytosolic fractions were prepared and analyzed by western blot using an anti-p47<i>^phox</i> antibody. (F) Cells were pretreated with PPADS (10 µM) or suramin (10 µM) for 1 h, and then treated with ATPγS for 15 min. The cytosolic and membrane fractions were prepared and analyzed by western blot using an anti-PKCα, anti-PKCι, or anti-PKCμ antibody. Data are expressed as mean±S.E.M. of three independent experiments. ^#<i>p</i><0.01, as compared with the cells exposed to ATPγS alone.</p

ATPγS induces PKC-dependent ROS generation in A549 cells.

<p>(A) Cells were labeled with DCF-DA (10 µM), pretreated with Gö6983 (10 µM), Gö6976 (10 µM), GF109203X (3 µM), Ro318220 (10 µM), or Rottlerin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. The fluorescence intensity (relative DCF fluorescence) was measured (gray bar). In addition, NADPH oxidase activity was determined (white bar). (B) Cells were pretreated with Gö6983 (10 µM), Gö6976 (10 µM), GF109203X (3 µM), Ro318220 (10 µM), or Rottlerin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. After incubation, ROS generation was determined by using CellROX™ Deep Red Reagent as described in Fig. 2E. (C) Cells were pretreated with Gö6983 (10 µM), Gö6976 (10 µM), GF109203X (3 µM), Ro318220 (10 µM), or Rottlerin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. The membrane and cytosolic fractions were prepared and analyzed by western blot using an anti-p47<i>^phox</i> antibody. Data are expressed as mean±S.E.M. of three independent experiments. ^#<i>p</i><0.01, as compared with the cells exposed to ATPγS alone.</p

ATPγS induces COX-2 expression via a cPLA₂/AA signaling.

<p>(A) Cells were treated with ATPγS for the indicated times. The expression of cPLA₂, COX-2, or COX-1 was determined by Western blot. (B) Cells were transfected with cPLA₂ or COX-2 siRNA, and then treated with ATPγS for 24 h or 6 h. The expression of cPLA₂ or COX-2 was determined by Western blot. (C) Cells were treated with AA for the indicated times. The expression of COX-2 or COX-1 was determined by Western blot. (D) Cells were treated with ATPγS or AA for the indicated times. The production of PGE₂ was measured. (E) Cells were transfected with cPLA₂ or COX-2 siRNA, and then treated with ATPγS for 6 h. The production of PGE₂ was measured. Data are expressed as mean±S.E.M. of three independent experiments. ^#<i>p</i><0.01, as compared with the cells exposed to vehicle alone (D) or scrambled siRNA+ATPγS (E).</p

Schematic diagram illustrating the proposed signaling pathway involved in ATPγS-induced COX-2 expression and PGE₂ generation in A549 cells.

<p>ATPγS activates the P2 receptor/PKC/NADPH oxidase pathway to enhance ROS generation, which in turn initiates the activation of Jak2 and STAT3, and ultimately induces COX-2-dependent PGE₂ generation in A549 cells.</p

ATPγS regulates COX-2 expression via PKCs in A549 cells.

<p>Cells were pretreated with Gö6983, Gö6976, Ro318220, or Rottlerin for 1 h, and then incubated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE₂ release. (C) Cells were treated with ATPγS (100 µM) for the indicated time intervals or PMA (1 µM) for 15 min. The cytosolic and membrane fractions were prepared and analyzed by western blot using an anti-PKCα, anti-PKCι, or anti-PKCμ antibody. β-actin and Gαs were used as a marker protein for cytosolic and membrane fractions, respectively. (D, E) Cells were transfected with siRNA of scrambled, PKCα, PKCι, or PKCμ, and then incubated with ATPγS for 6 h. The expression of PKCα, PKCι, PKCμ, and COX-2 were analyzed by western blot. The media were collected and analyzed for PGE₂ release. Data are expressed as mean±S.E.M. of three independent experiments. *<i>p</i><0.5; ^#<i>p</i><0.01, as compared with the cells exposed to ATPγS alone (A, B) or the cells transfected with scrambled siRNA and exposed to ATPγS alone (D, E).</p

ATPγS induces COX-2 expression via Jak2/STAT3 in A549 cells.

<p>Cells were pretreated with AG490 or CBE for 1 h, and then incubated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE₂ release. (C) Cells were treated with ATPγS (100 µM) for the indicated time intervals. The cell lysates were analyzed by western blot using an anti-phospho-Jak2, anti-phospho-STAT3, anti-STAT3, or anti-β-actin antibody. Cells were pretreated (D) without or (E) with AG490 (10 µM), CBE (10 µM), PPADS (10 µM), or suramin (10 µM) for 1 h, and then incubated with ATPγS (100 µM) for (D) the indicated time intervals or (E) 60 min. The cytosolic and nuclear fractions were prepared and analyzed by western blot using an anti-phospho-STAT3 or anti-STAT3 antibody. GAPDH and Lamin A were used as a marker protein for cytosolic and nuclear fractions, respectively. (F) Cells were transfected with siRNA of scrambled, Jak2, or STAT3, and then incubated with ATPγS for 6 h. The levels of Jak2, STAT3, and COX-2 expression were analyzed by western blot. The media were collected and analyzed for PGE₂ release. Data are expressed as mean±S.E.M. of three independent experiments. ^#<i>p</i><0.01, as compared with the cells exposed to ATPγS alone (A, B), the cells exposed to vehicle alone (C), or the cells transfected with scrambled siRNA and exposed to ATPγS alone (F, G).</p

Additional file 2: Table S1. of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

Phospho probabilities, process method, leading protein identifier, UniProt No. (if available), protein description, and protein functional class of the identified phosphopeptides (PDF 539 kb)

Additional file 1: of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

MS/MS Spectra. Spectra of the identified phosphopeptides (PDF 4714 kb)

Additional file 3: Figure S1. of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

The relative abundances of amino acids between −10 and +10 positions of the phosphorylated or non-phosphorylated His/Asp (PDF 377 kb)