1. CD26/DPPIV down-regulation in endometrial stromal cell migration in endometriosis 
Chin Wen Tan, B.Eng., Yie Hou Lee, Ph.D., Heng Hao Tan, M.B., B.S., Matthew Sie Kuei Lau, M.B., B.S., Mahesh Choolani, M.B., B.S., Ph.D., Linda Griffith, Ph.D., Jerry Kok Yen Chan, M.B., Ph.D. 
Fertility and Sterility 
Volume 102, Issue 1, Pages e9 (July 2014)
DOI: /j.fertnstert
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Migration and invasion under physiological oxygen tension in endometriosis. (A) Schematic flow chart of the study. (B) Oxygen tension in peritoneal cavity was evaluated by measuring peritoneal fluid %O2 in EM+ and EM− women. Fluid was collected at the beginning of the surgery and measured immediately by an automated blood gas analyzer. White = proliferative; gray = irregular; black = secretory; red = menses. Data were expressed as the mean ± SD. Migration (C) and invasion (D) of EM+ and EM− ESCs was examined under hypoxia (6.5% O2) or normoxia (20% O2). Cells were seeded in Oris Procell migration/invasion assay and were allowed to attach under hypoxic or normoxic condition for 2 hours (E). For invasion, collagen gel was coated onto the well after cell attachment. Seeded cells after 48 hours (F) were fixed and stained with crystal violet followed by measurement of the %covered area. Results were expressed as the mean ± SD (n = 7, 7). ∗P<.05; ∗∗P<.005; ∗∗∗P<.001; ∗∗∗∗P<.0005; $P<.05 as determined by paired t-test of same samples. EM+ = endometriosis; EM− = control; ESC = endometrial stromal cell; PCR = polymerase chain reaction. See text for other abbreviations.
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3. Figure 2
Gene expression (A, C) and protein levels (B, D, E) in cell culture supernatant in endometrial stromal cells under hypoxic condition. Pathway-focused polymerase chain reaction (PCR) array was used to quantify migration- and angiogenesis-related gene levels in messenger RNAs of cells derived from EM+ (n = 3) or EM− women (n = 3). Two genes, namely CXCL6 (A) and VEGFA (C), were selected from the microarray data. Proteins related to the gene results were quantified with their respective levels under hypoxic or normoxic condition. Results are expressed as the mean ± SD (n = 6, 6) for all ELISA data except SDF-1α (n = 7, 7). ∗P<.05, ∗∗P<.005, ∗∗∗P<.001, ∗∗∗∗P<.0005, #P<.05, ##P<.005 relative to EM−norm; %P<.05, %%P<.005 relative to EM−hyp as determined by Student's t-test; $P<.05, $$P<.005 as determined by paired t-test of same samples. EM+ = endometriosis; EM− = control. See text for other abbreviations.
Fertility and Sterility  , e9DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
(A) CD26/DPPIV gene expression in cell culture supernatant in endometrial stromal cells under hypoxia as determined by migration-related focused polymerase chain reaction (PCR) array. Messenger RNAs of cells were derived from EM+ (n = 3) or EM− women (n = 3). (B) CD26 surface marker expression in EM+ (n = 5) and EM− women (n = 4) when cultured under hypoxic condition. (C) CD26/DPPIV enzymatic activity was measured in cell lysates from EM+ (n = 6) or EM− (n = 6) endometrial stromal cells. ∗P<.05, ∗∗∗P<.001, ##P<.005 relative to EM−norm; %P<.05 relative to EM−hyp; ˆP<.05 relative to EM+norm as determined by Student's t-test; $P<.05, $$$P<.005 as determined by paired t-test of same samples. EM+ = endometriosis; EM− = control. See text for other abbreviations.
Fertility and Sterility  , e9DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
CD26/DPPIV involvement in migration (A) and invasion (B) of EM+ and EM− ESCs. Cells preincubated with or without diprotin A were seeded in Oris Procell migration/invasion assay and were allowed to attach under hypoxic or normoxic condition for 2 hours. Media supplemented with or without diprotin A, SDF-1α, or AMD3100 were applied to cover the cells. For invasion, collagen was coated onto the well after the attachment. Seeded cells after 24 hours were fixed and stained with crystal violet followed by measurement of the %covered area. Results are expressed as the mean ± SD (n = 7, 7). ∗P<.05, ∗∗P<.005, ∗∗∗P<.001, ∗∗∗∗P<.0005, $P<.05, $$P<.005 as determined by paired t-test of same samples. EM+ = endometriosis; EM− = control; ESC = endometrial stromal cell. See text for other abbreviations.
Fertility and Sterility  , e9DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Oxygen tension in peritoneal fluid (PF) and our cell culture system. (A) Oxygen tension measured in cell culture media being incubated in hypoxic or normoxic incubator for 1–3 days. (B) Oxygen tension in PF as categorized according to cycle phases of EM− (n = 12) and EM+ (n = 9) subjects. ####P<.0001 by Student's t-test. EM+ = endometriosis; EM− = control.
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Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

7. Supplemental Figure 2
Dose response of (A) SDF-1, (B) diprotin A, and (C) AMD3100 as determined by migration assay for 24 hours. #P<.05, ##P<.005, ###P<.001 relative to vehicle as determined by Student's t-test.
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Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

8. Supplemental Figure 3
Migration of endometrial stromal cells derived from EM+ (n = 4) or EM− women (n = 4) under different durations. ∗P<.05; ∗∗P<.005. EM+ = endometriosis; EM− = control.
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Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

9. Supplemental Figure 4
Effect of diprotin A on CD26 activity in endometrial stromal cells (ESCs) derived from EM+ (n = 3) or EM− women (n = 3). ∗P<.05, ∗∗P<.005, #P<.05, ##P<.005 relative to EM−norm (Student's t-test); $P<.05, $$P<.005 comparing difference brought by diprotin A (Student's t-test). EM+ = endometriosis; EM− = control.
Fertility and Sterility  , e9DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

10. Supplemental Figure 5
EM+ (n = 3) or EM− (n = 3) endometrial stromal cell (ESC) attachment without (A) or with (B) artificial extracellular matrix (ECM) coating. The ESCs were seeded onto plates coated with 10 μg/mL bovine serum albumin (BSA), collagen type I, fibronectin, and matrigel and allowed to adhere for 2 hours. Cells were then fixed and stained with 1% toluidine blue for 1 hour. After washing, the residual were solubilized in 2% sodium dodecyl sulfate (SDS) and the absorbance value was measured at 620 nm. Values were corrected for background binding to the respective wells. ∗P<.05; ∗∗P<.005. EM+ = endometriosis; EM− = control.
Fertility and Sterility  , e9DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions