FIG. 1. Effect of BDCM on the organization of desmosomal proteins in trophoblast cultures. Cytotrophoblast cells were incubated with or without BDCM under differentiation-inducing conditions (i.e., in KGM) for 48 h and then stained to reveal nuclei (red fluorescence) and desmosomal proteins (green fluorescence) as described in Methods. (A) Control nondifferentiated cytotrophoblast colonies. (B) Control differentiated syncytiotrophoblast-like colonies. The images in the other panels show trophoblasts cultured under differentiation-inducing conditions in the presence of BDCM. The concentrations were 2.0 mM (C); 0.2 mM (D); 0.02 mM (E) and 0.5 nM (F). The images are representative of samples from three separate experiments (cells from three different placentas). The horizontal bar represents 50 μm. From: Bromodichloromethane Inhibits Human Placental Trophoblast DifferentiationDisclaimer: The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Toxicol Sci. 2004;78(1):166-174. doi:10.1093/toxsci/kfh046 Toxicol Sci | Society of Toxicology

FIG. 2. Effect of BDCM on the formation of multinucleated trophoblast colonies. Cytotrophoblast cells were incubated with or without BDCM under differentiation-inducing conditions for 48 h. The data indicated by 0(K) and 0(H) represent control cultures incubated in KGM or HWM, respectively, in the absence of BDCM. The cultures were then stained to reveal nuclei and desmosomal proteins as described in Methods and Figure 1. Fluorescence images were captured, and the total numbers of nuclei were counted in random fields. At least 150 nuclei were counted in each case. The extent of multinucleated cell (i.e., syncytiotrophoblast) formation was calculated as described in Methods. Results are means ± SEM. from three separate experiments. Asterisks indicate values significantly different (p < 0.05) from untreated controls. Post-ANOVA trend analysis indicates a significant linear trend. From: Bromodichloromethane Inhibits Human Placental Trophoblast DifferentiationDisclaimer: The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Toxicol Sci. 2004;78(1):166-174. doi:10.1093/toxsci/kfh046 Toxicol Sci | Society of Toxicology

FIG. 3. Effect of BDCM on the secretion of immunoreactive and bioactive CG. Cytotrophoblasts were cultured for 48 h under differentiation-inducing conditions (i.e., in KGM) in the presence or absence of BDCM. The culture media were collected and analyzed for immunoreactive (A) and bioactive (B) CG as described in the Materials and Methods section. Results are means ± SEM from four separate experiments (four different placentas). The asterisks indicate values that are significantly different (p < 0.05) from the control. Post-ANOVA trend analysis indicates a significant linear trend for both datasets. From: Bromodichloromethane Inhibits Human Placental Trophoblast DifferentiationDisclaimer: The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Toxicol Sci. 2004;78(1):166-174. doi:10.1093/toxsci/kfh046 Toxicol Sci | Society of Toxicology

FIG. 4. Immunofluorescence localization of CG in trophoblasts exposed to BDCM. Cytotrophoblasts were cultured for 48 h under differentiation-inducing (KGM) conditions in the presence or absence of BDCM. The cultures were then fixed and stained using an anti-CG antibody as described in the Methods section. (A) Control cells incubated under non-differentiation-inducing conditions (B) Control cells incubated under differentiation-inducing conditions. The other images show cells incubated under differentiation-inducing conditions in the presence of different concentrations of BDCM. The concentrations used were 2.0 mM (C), 0.2 mM (D), 0.02 mM (E), 200 nM (F), and 0.5 nM (G). The last image (H) shows the immunoglobulin control. The horizontal bar represents 50 μm. From: Bromodichloromethane Inhibits Human Placental Trophoblast DifferentiationDisclaimer: The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Toxicol Sci. 2004;78(1):166-174. doi:10.1093/toxsci/kfh046 Toxicol Sci | Society of Toxicology

FIG. 5. Image analysis of CG immunofluorescence FIG. 5. Image analysis of CG immunofluorescence. Fluorescence micrographs from the experiment described in Figure 4 were imported into Image Pro software as described in Materials and Methods. Mean green fluorescence intensity values were obtained. Results are means ± SEMs from three separate experiments. At least 125 cell colonies were analyzed in each case. The data indicated by 0(K) and 0(H) represent control cultures incubated in KGM or HWM, respectively, in the absence of BDCM. The asterisks indicate values that are significantly different (p < 0.05) from the differentiated (K) control culture. ANOVA post-tests also showed a significant linear trend. From: Bromodichloromethane Inhibits Human Placental Trophoblast DifferentiationDisclaimer: The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Toxicol Sci. 2004;78(1):166-174. doi:10.1093/toxsci/kfh046 Toxicol Sci | Society of Toxicology

FIG. 6. Effect of BDCM on trophoblast viability FIG. 6. Effect of BDCM on trophoblast viability. Trophoblasts were cultured for 48 h under differentiation-inducing conditions in the presence or absence of BDCM. After removal of culture supernatants, the protein content of the adherent cells was measured (A). Culture supernatants were assayed for LDH activity (B) as described in Materials and Methods. Results are means ± SEM from three separate experiments. ANOVA showed no significant difference between means. From: Bromodichloromethane Inhibits Human Placental Trophoblast DifferentiationDisclaimer: The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Toxicol Sci. 2004;78(1):166-174. doi:10.1093/toxsci/kfh046 Toxicol Sci | Society of Toxicology