Figure 4.
Figure 4. PGE2 signaling regulates neutrophil apoptosis and NR4A expression in a PKA-dependent manner. (A) Ultrapurified neutrophils were cultured in the absence (red bars) or presence (blue bars) of Rp-8-Br-cAMPS (RP8) (0.7 mM) for 30 minutes prior to the addition of the following stimuli: N6/8-AHA (100 μM), PGE2 (10 μM), ATPγs [(1 μM), adenosine (100 μM), LPS (100 ng/mL), and GM-CSF (50 U/mL) for an additional 5 hours. Apoptosis was determined by light microscopy; data are expressed as means ± SEMs from 5 independent experiments. (B-C) In parallel experiments, neutrophils were cultured in the absence (red bars) or presence (blue bars) of Rp-8-Br-cAMPS (0.7 mM) for 30 minutes prior to the addition of the following stimuli: LPS (100 ng/mL), PGE2 (10 μM), adenosine (100 μM), or ATPγs (1 μM) for an additional 4 hours. (B) NR4A2 and (C) NR4A3 expression was measured by qPCR. Charts show the fold change from 0-hour control, where NR4A expression was normalized to the GAPDH loading control; data are from 3 independent experiments. Neutrophils were treated with media or (D) a concentration-response of PGE2 ranging from 10 nM to 10 μM or (E) butaprost (100 nM and 1 mM) in the presence (blue line) or absence (red line) of Rp-8-Br-cAMPS (0.7 mM) for 4 hours. (D-E) Apoptosis was determined by light microscopy; data are expressed as means ± SEMs from 3 independent experiments. Ultrapurified neutrophils were preincubated with media or Rp-8-Br-cAMPS (0.7 mM) for 30 minutes before the addition of media or PGE2 (10 μM) for an additional 1 or 4 hours. (F) NR4A2 and (G) NR4A3 expression was measured by qPCR. Charts show the fold change from 0-hour control, where NR4A expression was normalized to the GAPDH loading control; data are from 3 independent experiments. Data were analyzed by ANOVA with Bonferroni’s or Sidak’s posttest, and statistical differences are indicated by *P < .05, **P < .01, ***P < .001, and ****P < .0001. Comparisons were made between agonist alone or agonist plus Rp-8-Br-cAMPS treated conditions for panels D and E, or as the lines indicate for the other panels.

PGE2 signaling regulates neutrophil apoptosis and NR4A expression in a PKA-dependent manner. (A) Ultrapurified neutrophils were cultured in the absence (red bars) or presence (blue bars) of Rp-8-Br-cAMPS (RP8) (0.7 mM) for 30 minutes prior to the addition of the following stimuli: N6/8-AHA (100 μM), PGE2 (10 μM), ATPγs [(1 μM), adenosine (100 μM), LPS (100 ng/mL), and GM-CSF (50 U/mL) for an additional 5 hours. Apoptosis was determined by light microscopy; data are expressed as means ± SEMs from 5 independent experiments. (B-C) In parallel experiments, neutrophils were cultured in the absence (red bars) or presence (blue bars) of Rp-8-Br-cAMPS (0.7 mM) for 30 minutes prior to the addition of the following stimuli: LPS (100 ng/mL), PGE2 (10 μM), adenosine (100 μM), or ATPγs (1 μM) for an additional 4 hours. (B) NR4A2 and (C) NR4A3 expression was measured by qPCR. Charts show the fold change from 0-hour control, where NR4A expression was normalized to the GAPDH loading control; data are from 3 independent experiments. Neutrophils were treated with media or (D) a concentration-response of PGE2 ranging from 10 nM to 10 μM or (E) butaprost (100 nM and 1 mM) in the presence (blue line) or absence (red line) of Rp-8-Br-cAMPS (0.7 mM) for 4 hours. (D-E) Apoptosis was determined by light microscopy; data are expressed as means ± SEMs from 3 independent experiments. Ultrapurified neutrophils were preincubated with media or Rp-8-Br-cAMPS (0.7 mM) for 30 minutes before the addition of media or PGE2 (10 μM) for an additional 1 or 4 hours. (F) NR4A2 and (G) NR4A3 expression was measured by qPCR. Charts show the fold change from 0-hour control, where NR4A expression was normalized to the GAPDH loading control; data are from 3 independent experiments. Data were analyzed by ANOVA with Bonferroni’s or Sidak’s posttest, and statistical differences are indicated by *P < .05, **P < .01, ***P < .001, and ****P < .0001. Comparisons were made between agonist alone or agonist plus Rp-8-Br-cAMPS treated conditions for panels D and E, or as the lines indicate for the other panels.

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