Human uterine epithelial RL95-2 and HEC-1A cell-line adhesiveness: the role of plexin B1  Haggar Harduf, M.Sc., Shlomit Goldman, D.Sc., Eliezer Shalev, M.D.  Fertility and Sterility  Volume 87, Issue 6, Pages 1419-1427 (June 2007) DOI: 10.1016/j.fertnstert.2006.11.141 Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Attachment and growth of JAR spheroids in HEC-1A versus RL95-2 cells. (A) Representative photos of JAR spheroids cultured on a layer of HEC-1A and RL95-2 cells, after 3 days of incubation. Photos were captured by phase-contrast microscope at original magnification ×1,000 (B) Bar graph describes mean ± SEM of percent attachment after 24 hours of incubation of JAR spheroids to monolayer-cultured HEC-1A cells and to monolayer-cultured RL95-2 cells. Green bar, HEC-1A; blue bar, RL95-2. *P<.01. (C) Graph represents mean ± SEM of spheroid diameter in HEC-1A) green) and RL95-2 (blue). *P<.01. Harduf. Plexin-B1 in endometrial adhesiveness. Fertil Steril 2007. Fertility and Sterility 2007 87, 1419-1427DOI: (10.1016/j.fertnstert.2006.11.141) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Expression of plexin-B1 in endometrial cell lines HEC-1A versus RL95-2. (A) Representative Western blots from whole-cell extract. (B) Bar graph represents mean ± SEM of total plexin-B1 to GAPDH ratio from six independent experiments. Green bar, HEC-1A; blue bar, RL95-2. *P<.05. (C) Representative agarose gel of RT-PCR for plexin-B1 and GAPDH in HEC-1A and RL95-2 cells. Product size: plexin-B1, 150 bp; GAPDH, 240 bp. (D) Bar graph represents mean ± SEM of total plexin-B1 to GAPDH ratio from six independent experiments. Green bar, HEC-1A; blue bar, RL95-2. *P<.05. Harduf. Plexin-B1 in endometrial adhesiveness. Fertil Steril 2007. Fertility and Sterility 2007 87, 1419-1427DOI: (10.1016/j.fertnstert.2006.11.141) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Plexin-B1 expression level in HEC-1A-2-transfected cell line. (A) Representative agarose gel ′RT-PCR for plexin-B1 and GAPDH in RL95-2, HEC-1A, and HEC-1A-2 cells. Product size: plexin-B1, 150 bp; GAPDH, 240 bp. (B) Bar graph represents mean ± SEM of plexin-B1 to GAPDH ratio from four independent experiments. Green bar, HEC-1A; blue bar, RL95-2; azure bar, HEC-1A-2 cells. *P<.05. (C) Representative Western blots from whole-cell extract of RL95-2, HEC-1A, and HEC-1A-2 cells. (D) Bar graph represents mean ± SEM from five independent experiments of plexin-B1 to GAPDH ratio in HEC-1A, RL95-2, and HEC-1A-2. Green bar, HEC-1A; blue bar, RL95-2; azure bar, HEC-1A-2. *P<.05. (E) JAR spheroid attachment to HEC-1A-2 cells. Bar graph describes percent attachment (mean ± SEM) of 24-hour incubation of JAR spheroids to monolayer-cultured endometrial cells. Green bar, HEC-1A; blue bar, RL95-2; azure bar, HEC-1A-2. *P<.05. (F) Bar graph describes attachment percent (mean ± SEM) of 24-hour incubation of JAR spheroids to HEC-1A-2, treated with 10 ng/mL neutralizing antibodies against plexin-B1. Azure bar, nontreated HEC-1A-2 cells; striped bar, treated HEC-1A-2 cells. *P<.05. Harduf. Plexin-B1 in endometrial adhesiveness. Fertil Steril 2007. Fertility and Sterility 2007 87, 1419-1427DOI: (10.1016/j.fertnstert.2006.11.141) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Growth of JAR spheroids into HEC-1A-2 cells. (A) Representative photos of JAR spheroids cultured on a layer of RL95-2, HEC-1A, and HEC-1A-2 cells. JAR spheroids were labeled with membrane-permeable fluorescent dye CMFDA; photos were captured by fluorescence microscope, after 1 day of incubation, at original magnification ×1,000. (B) Graph represents mean ± SEM of spheroid diameter in HEC-1A (green), RL95-2 (blue), and HEC-1A-2 (azure). Harduf. Plexin-B1 in endometrial adhesiveness. Fertil Steril 2007. Fertility and Sterility 2007 87, 1419-1427DOI: (10.1016/j.fertnstert.2006.11.141) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions