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SILAC labeling coupled to shotgun proteomics analysis of membrane proteins of liver stem/hepatocyte allows to candidate the inhibition of TGF-beta pathway as causal to differentiation.

Montaldo C, Mancone C, Conigliaro A, Cozzolino AM, de Nonno V, Tripodi M - Proteome Sci (2014)

Bottom Line: By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation.Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways.Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute for Infectious Diseases L, Spallanzani, IRCCS, via Portuense 292, 00149 Rome, Italy. tripodi@bce.uniroma1.it.

ABSTRACT

Background: Despite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation.

Results: We apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation.

Conclusions: Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.

No MeSH data available.


Validation of the expression of selected cell surface proteins in RLSCs and DH. (A) Western blot analysis in total cell extract (WCE), soluble (S) and membrane (M) fractions for the indicated proteins. For each fraction, 10 μg of protein sample were loaded; GAPDH and Calnexin expressions were used respectively as total and membrane proteins loading control. One representative experiment out of three is shown. (B) Bands were analyzed by densitometry using Quality-One software (Bio-Rad laboratories, Richmond, CA). The Y axis shows the relative intensity respect to GAPDH (WCE) and calnexin (M). All data were from at least three independent experiments and shown as mean ± SD.
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Figure 2: Validation of the expression of selected cell surface proteins in RLSCs and DH. (A) Western blot analysis in total cell extract (WCE), soluble (S) and membrane (M) fractions for the indicated proteins. For each fraction, 10 μg of protein sample were loaded; GAPDH and Calnexin expressions were used respectively as total and membrane proteins loading control. One representative experiment out of three is shown. (B) Bands were analyzed by densitometry using Quality-One software (Bio-Rad laboratories, Richmond, CA). The Y axis shows the relative intensity respect to GAPDH (WCE) and calnexin (M). All data were from at least three independent experiments and shown as mean ± SD.

Mentions: Among the 57 proteins quantified, proteins associated to plasma membrane and with a measured fold of variation higher than 2.0 were considered for validation (Table 1). As shown in Figure 2, by means of western blotting analysis we found that the membrane expression levels of e-cadherin, integrin beta-4 and galectin-4 were similar to those measured by quantitative proteomics.


SILAC labeling coupled to shotgun proteomics analysis of membrane proteins of liver stem/hepatocyte allows to candidate the inhibition of TGF-beta pathway as causal to differentiation.

Montaldo C, Mancone C, Conigliaro A, Cozzolino AM, de Nonno V, Tripodi M - Proteome Sci (2014)

Validation of the expression of selected cell surface proteins in RLSCs and DH. (A) Western blot analysis in total cell extract (WCE), soluble (S) and membrane (M) fractions for the indicated proteins. For each fraction, 10 μg of protein sample were loaded; GAPDH and Calnexin expressions were used respectively as total and membrane proteins loading control. One representative experiment out of three is shown. (B) Bands were analyzed by densitometry using Quality-One software (Bio-Rad laboratories, Richmond, CA). The Y axis shows the relative intensity respect to GAPDH (WCE) and calnexin (M). All data were from at least three independent experiments and shown as mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4007997&req=5

Figure 2: Validation of the expression of selected cell surface proteins in RLSCs and DH. (A) Western blot analysis in total cell extract (WCE), soluble (S) and membrane (M) fractions for the indicated proteins. For each fraction, 10 μg of protein sample were loaded; GAPDH and Calnexin expressions were used respectively as total and membrane proteins loading control. One representative experiment out of three is shown. (B) Bands were analyzed by densitometry using Quality-One software (Bio-Rad laboratories, Richmond, CA). The Y axis shows the relative intensity respect to GAPDH (WCE) and calnexin (M). All data were from at least three independent experiments and shown as mean ± SD.
Mentions: Among the 57 proteins quantified, proteins associated to plasma membrane and with a measured fold of variation higher than 2.0 were considered for validation (Table 1). As shown in Figure 2, by means of western blotting analysis we found that the membrane expression levels of e-cadherin, integrin beta-4 and galectin-4 were similar to those measured by quantitative proteomics.

Bottom Line: By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation.Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways.Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute for Infectious Diseases L, Spallanzani, IRCCS, via Portuense 292, 00149 Rome, Italy. tripodi@bce.uniroma1.it.

ABSTRACT

Background: Despite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation.

Results: We apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation.

Conclusions: Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.

No MeSH data available.