Figure 1.
Co-localization of GFP expression with expression of the HSC marker desmin.
Immunohistochemistry was performed on sections of liver from collagen-GFP mice using antibodies to the HSC marker desmin (A) and GFP (B). Merged image shows colocalization of desmin and GFP expression (C). DAPI (blue) staining of nuclei is also shown in the merged image. Micrographs are at 40× magnification. Scale bars, 15 µm. Arrows point to a hepatic stellate cell. Insets show magnified view of one HSC.
Table 1.
Summary of differentially expressed genes.
Table 2.
KEGG Pathway Enrichment.
Figure 2.
mRNA and protein expression of markers of HSC activation.
(A) Relative mRNA expression levels of Col1a1, Acta2, Pdgfc, Tgfb, and Edn1 in AF-HSCs (solid bars) and GFP-HSCs (stripped bars) are shown. Values are normalized to 18S and given as the mean ±1 S.D. for 5 AF and 5 GFP HSC isolations. Significance was determined by a Student's t-test, *** p-value<0.001. ND means “not detected.” (B) Protein levels of ACTA2 in AF-HSCs and GFP-HSCs. Experiment was repeated with similar results.
Table 3.
Markers of HSC Activation.
Figure 3.
Total retinyl ester and triglyceride levels in liver and HSCs.
(A) Retinyl ester (RE) levels in livers from WT and collagen-GFP mice, expressed as µg RE per gram liver weight. (B) Retinyl ester levels in AF-HSCs and GFP-HSCs, expressed as µg RE per million cells. (C) Triglyceride (TG) levels in livers from WT and collagen-GFP mice, expressed as µg TG per gram liver weight. Mice were fasted for 4 hours prior to sacrifice and tissue collection. (D) TG levels in AF-HSCs and GFP-HSCs, expressed as µg TG per million cells. Significance was determined by a Student's t-test, * p-value<0.05. (E) Protein levels of LRAT in AF-HSCs and GFP-HSCs. Experiment was repeated with similar results.
Figure 4.
Assessment of lipid droplet content in AF-HSCs and GFP-HSCs after overnight culture.
AF-HSCs and GFP-HSCs were isolated by FACS and incubated at 37°C overnight on glass bottom dishes. Phase contrast and fluorescent images were captured at 60× magnification for both AF-HSCs (A) and GFP-HSCs (B). Scale bars represent 30 µm. Average number of lipid droplets (C), average diameter (µm) of lipid droplets (D) and total lipid droplet volume (µm3) (E) per HSC are shown. (F) Protein levels of ADRP in AF-HSCs and GFP-HSCs. Experiment was repeated with similar results.
Figure 5.
mRNA levels of retinoid- and lipid-related nuclear receptors.
Relative mRNA expression levels of Rxra, Rara, Rarg, and Ppara in AF-HSCs (solid bars) and GFP-HSCs (stripped bars) are shown. Values are normalized to 18S and given as the mean ±1 S.D. for 5 AF and 5 GFP HSC isolations.
Figure 6.
mRNA levels of retinol and fatty acid binding proteins.
Relative mRNA expression levels of Fabp4, Rbp4, and Rbp1 in AF-HSCs (solid bars) and GFP-HSCs (stripped bars) are shown. Values are normalized to 18S and given as the mean ±1 S.D. for 5 AF and 5 GFP HSC isolations.
Figure 7.
mRNA levels of lipid hydrolases.
Relative mRNA expression levels of Ces3 and LpL in AF-HSCs (solid bars) and GFP-HSCs (stripped bars) are shown. Values are normalized to 18S and given as the mean ±1 S.D. for 5 AF and 5 GFP HSC isolations. Significance was determined by a Student's t-test, * p-value<0.05 and ** p-value<0.01.
Table 4.
Retinoid- and Lipid-related Nuclear Receptors.
Table 5.
Retinol and Fatty Acid Binding Proteins.
Table 6.
Lipases.
Table 7.
Esterases.
Figure 8.
mRNA and protein expression of retinoid-metabolizing cytochrome P450 enzymes.
(A) Relative mRNA expression levels of Cyp2s1 and Cyp2e1 in AF-HSCs (solid bars) and GFP-HSCs (stripped bars) are shown. Values are normalized to 18S and given as the mean ±1 S.D. for 5 AF and 5 GFP HSC isolations. Significance was determined by a Student's t-test, ** p-value<0.01. (B) Protein levels of CYP2S1 and CYP2E1 in AF-HSCs and GFP-HSCs. Experiments were repeated with similar results.
Table 8.
Cytochrome P450 enzymes.
Figure 9.
Cyp2s1 is a retinoid-catabolizing CYP both highly expressed in HSCs and induced in hepatic fibrosis.
(A) HSCs and hepatocytes were isolated from male WT C57 mice at 3 months age. Relative mRNA expression levels of Cyp26a1, Cyp2e1, Cyp2c37, and Cyp2c39 (left panel) and Cyp26b1 and Cyp2s1 (right panel) are shown. (B) WT C57 male mice were given weekly injections of either corn oil or 0.5 ul CCl4/g B.W. administered in corn oil for 4 weeks and then sacrificed. Relative Cyp mRNA levels in whole liver homogenates were measured. Values are normalized to 18S and given as the mean ±1 S.D. Significance was determined by a Student's t-test, * p-value<0.05, ** p-value<0.01, *** p-value<0.001.