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1.
Figure 6

Figure 6. From: Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers.

Protein expression of Notch1, Jagged 1, and cleaved Notch1 in bipotential mouse embryonic liver cells as determined by Western blot analysis. Protein extracts from the 9A1 and 14B3 cells lines were grown in undifferentiated (basal) culture and differentiated as aggregates for 5 days. Jag1 and Notch1 proteins were present in both undifferentiated and differentiated cultures. The cleaved active form of Notch1, NICD, was present only in undifferentiated cells. α-Tubulin was used to ensure equal protein loading. Abbreviations: α-tub, α-tubulin; Jag1, Jagged 1; NICD, cleaved Notch1.

Scott A. Ochsner, et al. Stem Cells. ;25(10):2476-2487.
2.
Figure 3

Figure 3. From: Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers.

Heat map visualization of the 2,656 differentially regulated probe sets. Probe sets with an F p value less than .001 from the linear differential expression analysis of basal, Day 1, and Day 5 samples were clustered using DNA-Chip Analyzer. Hierarchical clustering of probe sets was done using Euclidean distance and average linkage. Each row has been standardized by subtracting the mean expression and dividing by the standard deviation. Five main probe-set expression patterns labeled A–E. Red indicates a higher relative expression and blue a lower relative expression.

Scott A. Ochsner, et al. Stem Cells. ;25(10):2476-2487.
3.
Figure 2

Figure 2. From: Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers.

Validation of microarray determined fold changes using reverse transcription (RT)-PCR. The expression of 34 genes (supplemental online Table 1) was assayed using RT-PCR in basal, D1 aggregate, and D5 aggregate bipotential mouse embryonic liver RNA samples. Log2 fold change values were calculated for the D1 versus basal and D5 versus basal contrasts. The RT-PCR determined fold change values for the 34 genes were plotted against the corresponding microarray fold change values from the mouse4302 microarray data. A best-fit line through all of the data points was calculated using a linear least squares method (lm function from the R stats package) and had an r2 value of .71; ● = D1 versus basal contrast; ○ = D5 versus basal contrast. Abbreviations: B, basal; D1, day 1; D5, day 5; FC, fold change; PCR, polymerase chain reaction.

Scott A. Ochsner, et al. Stem Cells. ;25(10):2476-2487.
4.
Figure 1

Figure 1. From: Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers.

Principal component and hierarchical clustering analysis with interquartile range filtered expression data. Screeplot from principal component analysis analyzing basal, Matrigel, and Day 5 aggregate bipotential mouse embryonic liver (BMEL) samples (A). The relative contribution of each principal component to the total experimental variance is shown. Scatter plot of principal component 1 versus principal component 2 (B). The biological replicates of the three BMEL culture conditions group together in distinct clusters separated along the PC1 axis. In each case, filled objects are from the 9A1 BMEL cell line, and open objects are from the 14B3 BMEL cell line. Hierarchical cluster display of BMEL samples using a 1 – Pearson correlation coefficient among samples as a distance measure and complete linkage agglomeration (C). Abbreviations: A, aggregate; B, basal; M, Matrigel; PC, principal component.

Scott A. Ochsner, et al. Stem Cells. ;25(10):2476-2487.
5.
Figure 4

Figure 4. From: Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers.

Protein expression of Stat3 and Stat5 in bipotential mouse embryonic liver (BMEL) cells as determined by Western analysis. Nuclear extracts from the 9A1 BMEL cell line grown under basal, Day 1, and Day 5 culture conditions were assayed for both total and phosphorylated stat protein (A). Both Stat5 and Stat3 phosphorylation ([A], panels 1 and 3) as well as total Stat5 and Stat3 protein levels are shown ([A], panels 2 and 4). Each lane is an independent biological replicate. Nuclear liver extracts from newborn mice treated with LPS (5 mg/kg) or vehicle (untreated) for 1 hour serve as a control for the size and phosphorylation of Stat3 and Stat5 relative to the basal samples (B). Again, both Stat5 and Stat3 phosphorylation ([B], panels 1 and 3) as well as total Stat5 and Stat3 protein levels are shown ([B], panels 2 and 4). The sizes of the Stat5 and Stat3 bands in the basal samples are the same as those induced by LPS in newborn mice. Abbreviation: LPS, lipopolysaccharide; MW, molecular weight; Unt, untreated.

Scott A. Ochsner, et al. Stem Cells. ;25(10):2476-2487.
6.
Figure 5

Figure 5. From: Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers.

Electromobility shift assay (EMSA) for Stat3 and Stat5 proteins. Stat5 EMSA in bipotential mouse embryonic liver (BMEL) nuclear extracts (A). Lanes 4–12 do not show any detectable Stat5 binding to a rat Csn2 oligonucleotide probe in samples from BMEL cells cultured under basal, Day 1, or Day 5 aggregate conditions (A). Extracts from an adult untreated liver ([A], lanes 1–3) serve as a control for Stat5 protein binding to the rat Csn2 oligonucleotide. Stat5 binding is specific as a Stat5 antibody causes a supershift ([A], lane 2) and 100× nonlabeled rat Csn2 oligonucleotide competes ([A], lane 3) with labeled rat Csn2 for protein binding. Stat3 EMSA in BMEL nuclear extracts (B). Lanes 7–15 do not show any detectable Stat3 binding to a mouse Icam1 oligonucleotide probe using the same BMEL cell extracts used in (A). Extracts from newborn animals treated with LPS (5 mg/kg) ([B], lanes 4–6) serve as a control for Stat3 protein binding to the mouse Icam1 oligonucleotide. Stat3 binding is specific as a Stat3 antibody ([B], lane 5) causes a supershift and 100× nonlabeled mouse Icam1 oligonucleotide competes ([B], lane 6) with labeled mouse Icam1 for protein binding. Solid arrows point to specific Stat5 and Stat3 protein binding, asterisks show supershifted band(s), and a., b., and c. indicate unidentified specific protein binding to the mouse Icam1 oligonucleotide that does not supershift with the Stat3 antibody. Abbreviations: Ab., probe + antibody; Comp., probe + 100× nonlabeled oligonucleotide; Csn2, rat casein beta oligonucleotide; Icam, mouse intercellular adhesion molecule oligonucleotide; L:LPS, newborn animals treated with lipopolysaccharide; UL, untreated newborn animals.

Scott A. Ochsner, et al. Stem Cells. ;25(10):2476-2487.
7.
Figure 7

Figure 7. From: Transcriptional Profiling of Bipotential Embryonic Liver Cells to Identify Liver Progenitor Cell Surface Markers.

Detection of candidate cell surface markers in livers chemically induced to undergo progenitor cell proliferation. Mice were treated with DDC for 3 weeks to induce hepatic damage, atypical ductular reaction, and oval cell expansion. Routine H&E staining of liver sections taken from animals treated with DDC (A). Regions staining dark purple correspond to regions of DDC-induced ductular proliferation containing oval cells and hematopoietic infiltrate (solid arrows). The pink staining areas surrounding regions of ductular proliferation correspond to hepatocytes and endothelial-lined sinusoids. Cytokeratin 19 immunostaining of normal adult liver and DDC-treated liver, respectively, illustrates the extent of the DDC-induced ductular reaction (B, C). In situ localization of Cd24a mRNA using sense and antisense cDNA probes (D–F). Hybridization using a control sense probe (D). Hybridization using an antisense probe at low and high magnifications where arrows point to regions of digoxigenin-labeled anti-sense probe/antibody precipitate (E, F). Low and high magnifications of cytokeratin 19 immunostaining in animals treated with DDC (G, J). Cd24a immunostaining of the same tissue sections from panel (G) and panel (J) (H, K). The merge of these photomicrographs illustrated a clear expression overlap between these two proteins in regions of ductular reaction (I, L). Cd24a and Cd45 immunostaining of a ductular reaction region from animals treated with DDC (M–O). Merging areas of CD24a and Cd45 staining showed that these two proteins are not expressed on the same cells, indicating that cells expressing Cd24a are not derived from hematopoietic cells (O);*, areas of Cd24a immunostaining that do not overlap with cytokeratin 19 immunostaining. Abbreviations: DDC, diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate; dr, diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate-induced ductular reaction; h, hepatocytes; wks, weeks.

Scott A. Ochsner, et al. Stem Cells. ;25(10):2476-2487.

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