JCS019315 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Table S2 -

  Adobe PDF

• Supplemental Table S3 -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Ep-CAM staining in sorted cells. (a) Ep-CAM-positive fraction showed Ep-CAM staining (red) in 84±6% cells. (b) Ep-CAM-negative fraction showed few Ep-CAM stained cells, 9±7%, P<0.001. (c) Negative control without primary antibody showed no staining. Nuclei counterstained with DAPI. Original magnification ×200. Cytospun cells were fixed in 4% paraformaldehyde in PBS for 10 minutes and blocked with 3% goat serum in PBS for 1 hour before anti-EpCAM (MCA850H, Serotec Ltd. Raleigh, NC) for 1 hour at room temperature. After three washes with PBS for 10 minutes each, Rhodamine-conjugated mouse-specific goat IgG (1:300; #715-295-150, Jackson Immuno Research, West Grove, PA) was added for 1 hour.

• Supplemental Figure S2 -

  Fig. S2. Immunohistochemical staining for specific liver markers in Ep-CAM-positive cells. Freshly isolated Ep-CAM-positive fetal liver cells with staining for hepatic and biliary markers as indicated. Most cells expressed hepatic and biliary markers. Quantitative analysis of gene expression is given in Table 4. Original magnification ×200. Histochemical staining for the hepatobiliary markers, glycogen, dipeptidyl peptidase IV (DPPIV), γ-glutamyltranspeptidase (GGT) and glucose-6-phosphatase (G-6-P) utilized published protocols. To colocalize glycogen and cytokeratin (CK)-19, cells were first immunostained for CK-19 and then stained for glycogen content.

• Supplemental Figure S3 -

  Fig. S3. Phenotypic perturbation in cultured fetal cells. Panels a, b and middle panel in d show primary cultures (P0) and panels c and d show cells passaged thrice (P3). Panels a-c, left, show albumin staining with green FITC signal. Middle panels in a-c show vimentin or CK-19 staining (red, Rhodamine), whereas panels on right show merged images. Panel d on left shows expression of α-smooth muscle actin (red). Middle and right panels in d show cell proliferation with Ki-67 immunostaining using DAB for visualization. DAPI nuclear counterstain, blue color, all other panels. Original magnification ×200.

• Supplemental Figure S4 -

  Fig. S4. Efficiency of LV-transduction in cultured fetal cells. Panels a, b and c show cells passaged once (P1) with trznsduction using LVs expressing GFP under PGK, Alb and TTR promoters, respectively. The panels show DAPI staining on left to visualize cell nuclei, GFP staining in the middle to demonstrate expression of the transgene and merged images on the right to show the extent of cell transduction. The majority of cells were transduced under the conditions used. Original magnification ×200.

• Supplemental Figure S5 -

  Fig. S5. Differential regulation of vimentin and α-SMA expression in cultured fetal liver cells. The panels show DAPI staining of nuclei (a) and immunostaining for vimentin (b) or α-SMA (c) with merged images (d) to demonstrate that although vimentin was expressed in all cells, α-SMA was expressed in only some cells. Original magnification ×200

• Supplemental Figure S6 -

  Fig. S6. Microarray analysis showing changes in HGF, EGF/TGF-α and PDGF signaling. The data were obtained by PathwayStudio analysis and represent genes expressed better in PP cells (pink) or less well in PP cells (green). Boxes in grey indicate genes that were not in the query lists of differentially expressed genes. Panel a shows regulation of several critical members transducing HGF signaling in cultured cells compared with PP cells. These included c-Met and phospholipase C gamma 2 (PLCG2) receptors, inositol phosphate-5-phosphatase D (INPP5D), the adapter molecule GRB2 (growth factor receptor bound 2), the docking protein GAB1 (GRB2-associated binding protein1), which recruits phosphatidylinositol-3 kinase, STAT3 (signal tranducer and activator of transcription-3), which directs the acute phase response, and WASL (Wiskott-Aldrich syndrome-like) gene, which can regulate actin polymerization. Similarly, as shown in Panel b, TGF-α and EGF/TGF-α receptor were regulated during cell culture with perturbations in STAT3 expression. By contrast, as shown in Panel c, PDGF signaling seemed to be mostly unperturbed in fetal cells.

• Supplemental Figure S7 -

  Fig. S7. Regulation of FGF receptor pathways in fetal liver cells. These data demonstrate the overall regulation of FGF receptor pathways in PP cells vs cultured cells. Genes expressed better in PP cells are in pink, those expressed less well in PP are in green, and those not represented in the lists are in grey. Panel a shows that FGFR1 was expressed better in PP cells, although in contrast with P0 cells, FGF-1 and FGF-7 were expressed at lower levels in P1 and P3-6 cells. By contrast, FGFR2 and FGFR3 were less well expressed in PP cells. FGFR2 signaling appeared to be associated with superior expression of the downstream EGR2 (early growth response-2) gene in cultured cells versus PP cells.

• Supplemental Figure S8 -

  Fig. S8. Cell cycle profiles during various manipulations. The data are from P3 fetal cells shown in main Fig. 8. The flow cytometric profiles of cells with FBS (control) or after treatment with HGF, OSM or no serum were similar. These findings excluded the possibility that altered cell proliferation confounded changes in gene expression under these conditions.