1. Arachidonic acid regulation of the cytosolic phospholipase A2α/cyclooxygenase-2 pathway in mouse endometrial stromal cells 
Zhen-Ao Zhao, Ph.D., Zhi-Rong Zhang, B.S., Xiu Xu, Ph.D., Wen-Bo Deng, B.S., Ming Li, B.S., Jing-Yu Leng, B.S., Xiao-Huan Liang, Ph.D., Zeng-Ming Yang, Ph.D. 
Fertility and Sterility 
Volume 97, Issue 5, Pages e9 (May 2012)
DOI: /j.fertnstert
Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Arachidonic acid (AA) stimulates cytosolic phospholipase A2α (cPLA2α) phosphorylation through ERK1/2 and p38 MAPKs. (A) Western blots showing effects of AA on the phosphorylation of cPLA2α, p38, ERK1/2, and JNK. After 24 hours of serum deprivation, mouse endometrial stromal cells were treated with AA (15 μmol/L) for the indicated lengths of time. (B) Effects of MAPK inhibitors on cPLA2α phosphorylation. Serum-starved stromal cells were pretreated with p38 inhibitor (SB203580, 5 μmol/L), MEK inhibitor (U0126, 5 μmol/L), or JNK inhibitor (SP600125, 2 μmol/L) for 1 hour and then treated with AA (15 μmol/L) for 6 hours. Each experiment was repeated three times.
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3. Figure 2
Arachidonic acid (AA) regulates cyclooxygenase-2 (COX-2) expression through p38, ERK1/2, and C/EBPβ. (A) Effects of AA on C/EBPβ protein expression and phosphorylation. (B) Effects of MAPK inhibitors on COX-2 protein expression, C/EBPβ expression, and C/EBPβ phosphorylation induced by AA. Serum-starved stromal cells were treated with AA (15 μmol/L) and one of the following inhibitors for 1 hour: 5 μmol/L SB203580, 5 μmol/L U0126, or 2 μmol/L SP (C) Validation on effects of MAPK inhibitors on Cox-2 mRNA expression. Data are expressed as mean ± SEM. ∗P<.05. (D) COX-2 expression was diminished by siRNA against C/EBPβ. After transfection for 24 hours, stromal cells was treated with AA (15 μmol/L) for 6 hours. (E) COX-2 expression was diminished by inhibitory C/EBPβ (LIP) transfection. After LIP transfection for 24 hours, stromal cells were treated with AA (15 μmol/L) for 6 hours. Phospho-C/EBPβ (Thr188) antibody was used to detect LIP expression.
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4. Figure 3
Luciferase assay showing Cox-2 promoter activity induced by C/EBPβ. After cotransfection of C/EBPβ, Cox-2 promoter reporter, and pRL-SV40 (internal reference) for 24 hours, stromal cells were lysed for luciferase assay. The mutated sequences are underlined. Circle indicates mutated site. Data are expressed as mean ± SEM. ∗P<.05.
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5. Figure 4
Arachidonic acid (AA) regulates the cPLA2α/COX-2 pathway independently from prostaglandin biosynthesis. (A) Real-time reverse-transcription PCR of GDF15 mRNA after uterine stromal cells were treated with 2, 10, and 20 μmol/L indomethacin for 1 hour. (B) AA up-regulated cPLA2α/COX-2 pathway in the presence of COX inhibitor. Serum-starved stromal cells were pretreated with indomethacin (10 μmol/L) for 1 hour and then treated with AA (15 μmol/L) for 6 hours. (C) Effects of AA analogue ETYA on the cPLA2α/COX-2 pathway. Serum-starved stromal cells were treated with ETYA (15 μmol/L) for 6 hours. ∗P<.05.
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6. Supplemental Figure 1 Quantification analyses of Figure 1. ∗P<.05.
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7. Supplemental Figure 2
Quantification analyses of Figure 2A and B. ∗P<.05.
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8. Supplemental Figure 3
COX-2 expression is not regulated by PPARδ. (A) Effects of GW on Cox-2 mRNA expression. After 24 hours of serum deprivation, mouse endometrial stromal cells were treated with increasing concentrations of GW (1–1,000 nmol/L) for 6 hours. (B) Effect of PPARδ overexpression on COX-2 protein level. Stromal cells were transfected with PPARδ overexpression vector for 24 hours. COX-2 and PPARδ protein were detected by Western blot. (C) Effect of siRNA against PPARδ on PPARδ mRNA level. Stromal cells were transfected with siRNA against PPARδ for 24 hours. PPARδ mRNA was detected by real-time reverse-transcription (RT) PCR. (D) Effects of siRNA against PPARδ on Cox-2 mRNA level. Stromal cells were transfected with siRNA against PPARδ for 24 hours. Cox-2 mRNA was detected by real-time RT-PCR. Data are expressed as mean ± SEM. ∗P<.05.
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9. Supplemental Figure 4
AA-induced Cox-2 expression is independent from NFκB. (A) AA had no effects on IκB phosphorylation. After 24 hours of serum deprivation, mouse endometrial stromal cells were treated with AA (15 μmol/L) for indicated lengths of time. (B) AA-induced Cox-2 expression was not inhibited by IKK inhibitor Bay Serum-starved stromal cells were pretreated with Bay (1 μmol/L) for 1 hour, and then treated with AA (15 μmol/L) for 6 hours. P<.05.
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10. Supplemental Figure 5
Quantification analyses of Figure 2D and E. ∗P<.05.
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11. Supplemental Figure 6
PGE2 and PGI2 regulation on COX-2 expression. Mouse uterine stromal cells from day 4 uteri were treated with 0, 5, 10, and 20 μmol/L PGE2 or 0, 1, 5, and 10 μmol/L cPGI2 for 6 hours. COX-2 expression was determined by real-time reverse-transcription PCR. ∗P<.05.
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12. Supplemental Figure 7
Signaling pathway of AA regulation of cPLA2α/COX-2.
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