Figure legends

Figure 1: Distribution of p97/VCP in the early human placenta (4th-7th week). Tissue sections were taken from the 4th, 5th, 6th and the 7th week of first trimester human placenta. (a) Cytotrophoblast cells (CT) in mesenchymal villi (MV) (4th week) were strongly immunopositive for p97/VCP whereas syncytiotrophoblast cells (SN) were only barely immunopositive. Vascular endothelium (arrow) was moderately stained. Some of the hematopoietic cells (double arrows) were also immunopositive. (b) The cytoplasm of cell columns (CC) was weakly immunopositive for p97/VCP although cytotrophoblast cells (CT) were strongly positive in immature intermediate villi (IMIV) (5th week). (c) Hofbauer cells (H) showed strong immunoreactivity for p97/VCP in IMIV (6th week). The endothelium (V) showed no or weak immunoreactivity. Some of the stromal cells (arrowheads) stained moderately. No staining was seen in angiogenic cell cords (ACC). (d) The HSCORE of p97/VCP immunostaining intensities in early human placental villi. The data are represented as mean + SEM. a: p < 0.05, 4th week vs.6th and 7th week, b: 5th week vs. 7th week. Scale bars represent 50 m (a) and 100 m (b and c).

Figure 2: Distribution of p97/VCP in the early human placenta (6th and 7th week). (a) Differently shaped and sized Hofbauer cells (H) are observed in IMIV. Large cytoplasmic vacuoles and the exentric nucleus of Hofbauer cells are found to be immunopositive (upper and lower insets). (b) Cytoplasmic vacuoles of a Hofbauer cell but not the nucleus are positive for p97/VCP (lower inset). No staining was observed in negative controls (upper inset). IMIV = immature intermediate villus, Scale bars represent 100 m (a) and 50 m (b).

Figure 3: Distribution of CSN5 in the early human placental villi. (a-c) CSN5 was localized in cytotrophoblasts (CT), syncytiotrophoblasts (SN), Hofbauer cells (H), vascular endothelium (V) and stromal cells (arrowheads) in IMIV (4th-7th weeks). No staining was seen in negative controls (d, inset).

The cytoplasm of Hofbauer cells was immunopositive for CSN5 (inset in a). Scale bars represent 100 m. (d) HSCORE intensities of CSN5 immunostaining in early human placental villi. Data are represented as mean + SEM. a: p< 0.05, 4th week vs.7th week, b: 5th week vs. 7th week.

Figure 4: Western blot analysis of p97/VCP (a) and CSN5 (b) in the first trimester of human placenta. p97/VCP (97 kDa) and CSN5 (38 kDa) were detected by western blotting. -actin (43 kDa) was used as loading control. Immunoblot bands were quantified by an optical densitometer. The OD (optical density) values of p97/VCP and CSN5 bands were normalized to the OD values of -actin bands. The data in the graphs are presented as means SEM.

Figure 5: CSN5 interacts with p97/VCP in the 6th week of human placenta. (a) Lysates from early human placenta (input, lane 1) were used for immunoprecipitation (IP) with anti-p97/VCP antibody (lane 2) and isotype control antibody (Ctrl., lane 3) and analyzed by immunoblotting (IB) for CSN5 and p97/VCP. (b) Reciprocal IP was performed using anti-CSN5antibody (lane 2). Immunoprecipitates were immunoblotted for the detection of p97/VCP and CSN5.