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HEP_22880_sm_SuppFig1.TIF334KSupplementary S1. S1a) Silver staining of protein lysates from hepatocytes obtained with different lysis buffers. Cell cultured on CM or CS were lysed using either ice cold RIPA plus shear force, RIPA plus sonication, or a commercial lysis buffer from Roche. 30 μg of soluble protein were resolved in 4–12% SDS/PAGE gel and stained with silver staining kit. S1b) Western blot analysis of Smad2 phosphorylation in CM or CS cultured hepatocytes. Cell lysates from CS cultured hepatocytes stimulated with TGF-β were obtained with different lysis protocols as described in A). A positive control from CM cultured hepatocytes stimulated with TGF-β for 1h was included. Smad2 was used as loading control
HEP_22880_sm_SuppFig2.TIF142KSupplementary S2. Western blot analysis of phosphor-tyrosin and total FAK of immunoprecipitated FAK. Protein lysates were obtained from CM cultured hepatocytes with RIPA buffer. A non-related IgG was used as isotype control for immunoprecipitation. IgG heavy chain signal was used as loading control.
HEP_22880_sm_SuppFig3.TIF917KSupplementary S3. Infection rate analysis in hepatocytes cultured in CS. Hepatocytes were infected with AdGFP on day 0. Pictures of phase contrast and fluorescence were taken 24h later.
HEP_22880_sm_SuppFig4a.TIF60KSupplementary S4. S4a–c: Real time PCR analysis of Snail, ZEB1 and claudin-1 (EMT markers), in hepatocytes cultured in CM or CS, untreated or stimulated with 5 ng/mL TGF-β for 24 or 48h.
HEP_22880_sm_SuppFig4b.TIF59KSupplementary S4. S4a–c: Real time PCR analysis of Snail, ZEB1 and claudin-1 (EMT markers), in hepatocytes cultured in CM or CS, untreated or stimulated with 5 ng/mL TGF-β for 24 or 48h.
HEP_22880_sm_SuppFig4c.TIF68KSupplementary S4. S4a–c: Real time PCR analysis of Snail, ZEB1 and claudin-1 (EMT markers), in hepatocytes cultured in CM or CS, untreated or stimulated with 5 ng/mL TGF-β for 24 or 48h.
HEP_22880_sm_SuppFig4d.TIF59KSupplementary S4. S4a–c: Real time PCR analysis of Snail, ZEB1 and claudin-1 (EMT markers), in hepatocytes cultured in CM or CS, untreated or stimulated with 5 ng/mL TGF-β for 24 or 48h.
HEP_22880_sm_SuppFig5a.TIF950KSupplementary S5. S5a–b Bile canaliculi secretion of CMFDA. CMFDA was added to hepatocytes plated on either CM, CM+CG, CG or CS. Fluorecent and phase contrast pictures were taken 30 min later. Active bile canaliculi secrete the fluorescent product of CMFDA hydrolysis. S5c: Quantification of bile canaliculi per field 200X magnification. Representative of 3 independent fields.
HEP_22880_sm_SuppFig5b.TIF695KSupplementary S5. S5a–b Bile canaliculi secretion of CMFDA. CMFDA was added to hepatocytes plated on either CM, CM+CG, CG or CS. Fluorecent and phase contrast pictures were taken 30 min later. Active bile canaliculi secrete the fluorescent product of CMFDA hydrolysis. S5c: Quantification of bile canaliculi per field 200X magnification. Representative of 3 independent fields.
HEP_22880_sm_SuppFig5c.TIF58KSupplementary S5. S5a–b Bile canaliculi secretion of CMFDA. CMFDA was added to hepatocytes plated on either CM, CM+CG, CG or CS. Fluorecent and phase contrast pictures were taken 30 min later. Active bile canaliculi secrete the fluorescent product of CMFDA hydrolysis. S5c: Quantification of bile canaliculi per field 200X magnification. Representative of 3 independent fields.
HEP_22880_sm_SuppFig6a.TIF89KSupplementary S6. Dedifferentiation is an adaptive process that requires transcriptional and translational activities. Microarray analysis of gene expression comparing hepatocytes cultured in CM on day 2 to day 1. S6a) Over represented gene ontology clusters (GO) of genes modulated during cultivation in CM S6b) Effect of protein and mRNA synthesis inhibition in spontaneous EMT on CM. Factin stress fiber formation analyzed as in B. Active hydrolysis of CMFDA esters indicates live cells.
HEP_22880_sm_SuppFig6b.TIF1129KSupplementary S6. Dedifferentiation is an adaptive process that requires transcriptional and translational activities. Microarray analysis of gene expression comparing hepatocytes cultured in CM on day 2 to day 1. S6a) Over represented gene ontology clusters (GO) of genes modulated during cultivation in CM S6b) Effect of protein and mRNA synthesis inhibition in spontaneous EMT on CM. Factin stress fiber formation analyzed as in B. Active hydrolysis of CMFDA esters indicates live cells.
HEP_22880_sm_SuppFig7a.TIF1408KSupplementary S7. Extracellular matrix composition and structure effect on polarity and sensitivity to apoptosis. S7a–b) Phase contrast images of hepatocyes cultured on dry stiff collagen (CM), on a single layer of collagen gel (CG), between a layer of stiff and gel collagen (CM+CG) or two layers of collagen gel (CS). The cells were stimulated by 5 ng/mL TGF-β for 48h. EMT is observed by spindle-shape of hepatocytes. Apoptotic bodies (arrows) are observed as in Fig 3A. S7c) Quantification of apoptotic bodies as index of apoptosis. S7d) Apoptosis induction as evidenced by Western blot analysis of PARP (full length and cleaved), active caspase-3 and Bcl-2. GAPDH was used as loading control.
HEP_22880_sm_SuppFig7b.TIF1084KSupplementary S7. Extracellular matrix composition and structure effect on polarity and sensitivity to apoptosis. S7a–b) Phase contrast images of hepatocyes cultured on dry stiff collagen (CM), on a single layer of collagen gel (CG), between a layer of stiff and gel collagen (CM+CG) or two layers of collagen gel (CS). The cells were stimulated by 5 ng/mL TGF-β for 48h. EMT is observed by spindle-shape of hepatocytes. Apoptotic bodies (arrows) are observed as in Fig 3A. S7c) Quantification of apoptotic bodies as index of apoptosis. S7d) Apoptosis induction as evidenced by Western blot analysis of PARP (full length and cleaved), active caspase-3 and Bcl-2. GAPDH was used as loading control.
HEP_22880_sm_SuppFig7c.TIF61KSupplementary S7. Extracellular matrix composition and structure effect on polarity and sensitivity to apoptosis. S7a–b) Phase contrast images of hepatocyes cultured on dry stiff collagen (CM), on a single layer of collagen gel (CG), between a layer of stiff and gel collagen (CM+CG) or two layers of collagen gel (CS). The cells were stimulated by 5 ng/mL TGF-β for 48h. EMT is observed by spindle-shape of hepatocytes. Apoptotic bodies (arrows) are observed as in Fig 3A. S7c) Quantification of apoptotic bodies as index of apoptosis. S7d) Apoptosis induction as evidenced by Western blot analysis of PARP (full length and cleaved), active caspase-3 and Bcl-2. GAPDH was used as loading control.
HEP_22880_sm_SuppFig7d.TIF318KSupplementary S7. Extracellular matrix composition and structure effect on polarity and sensitivity to apoptosis. S7a–b) Phase contrast images of hepatocyes cultured on dry stiff collagen (CM), on a single layer of collagen gel (CG), between a layer of stiff and gel collagen (CM+CG) or two layers of collagen gel (CS). The cells were stimulated by 5 ng/mL TGF-β for 48h. EMT is observed by spindle-shape of hepatocytes. Apoptotic bodies (arrows) are observed as in Fig 3A. S7c) Quantification of apoptotic bodies as index of apoptosis. S7d) Apoptosis induction as evidenced by Western blot analysis of PARP (full length and cleaved), active caspase-3 and Bcl-2. GAPDH was used as loading control.
HEP_22880_sm_SuppFig8a.TIF64KSupplementary S8. Densitometric quantification of phosphor-Smad2 (S8a), phosphor-p38 (S8b), and phospho-JNK (S8c) relative to GAPDH, from CM cultured hepatocytes, in 0,1% DMSO or in the presence of the indicated inhibitors. Stimulations were done for 48h with 5 ng/mL TGF-β.
HEP_22880_sm_SuppFig8b.TIF60KSupplementary S8. Densitometric quantification of phosphor-Smad2 (S8a), phosphor-p38 (S8b), and phospho-JNK (S8c) relative to GAPDH, from CM cultured hepatocytes, in 0,1% DMSO or in the presence of the indicated inhibitors. Stimulations were done for 48h with 5 ng/mL TGF-β.
HEP_22880_sm_SuppFig8c.TIF60KSupplementary S8. Densitometric quantification of phosphor-Smad2 (S8a), phosphor-p38 (S8b), and phospho-JNK (S8c) relative to GAPDH, from CM cultured hepatocytes, in 0,1% DMSO or in the presence of the indicated inhibitors. Stimulations were done for 48h with 5 ng/mL TGF-β.
HEP_22880_sm_SuppFig9a.TIF331KSupplementary S9. S9a: Western blot analysis of phospho-Smad2, phospho-p38 and phospho-ERK in AML12 cells, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9b) Western blot analysis of phospho-Smad2 and phospho-p38 in hepatocytes cultured in either CM, CG or CS, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9c) Densitometric quantification of p38 phosphorylation relative to β-actin, from hepatocytes treated as described in S9b.
HEP_22880_sm_SuppFig9b.TIF223KSupplementary S9. S9a: Western blot analysis of phospho-Smad2, phospho-p38 and phospho-ERK in AML12 cells, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9b) Western blot analysis of phospho-Smad2 and phospho-p38 in hepatocytes cultured in either CM, CG or CS, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9c) Densitometric quantification of p38 phosphorylation relative to β-actin, from hepatocytes treated as described in S9b.
HEP_22880_sm_SuppFig9c.TIF61KSupplementary S9. S9a: Western blot analysis of phospho-Smad2, phospho-p38 and phospho-ERK in AML12 cells, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9b) Western blot analysis of phospho-Smad2 and phospho-p38 in hepatocytes cultured in either CM, CG or CS, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9c) Densitometric quantification of p38 phosphorylation relative to β-actin, from hepatocytes treated as described in S9b.
HEP_22880_sm_SuppFig9d.TIF203KSupplementary S9. S9a: Western blot analysis of phospho-Smad2, phospho-p38 and phospho-ERK in AML12 cells, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9b) Western blot analysis of phospho-Smad2 and phospho-p38 in hepatocytes cultured in either CM, CG or CS, stimulated with TGF-β for the indicated times. β-actin was used as loading control. S9c) Densitometric quantification of p38 phosphorylation relative to β-actin, from hepatocytes treated as described in S9b.
HEP_22880_sm_SuppFig10a-b.TIF726KSupplementary S10. S10a) TGF-β-induced apoptosis in AML12 cells was detected by genomic DNA fragmentation. Cells were stimulated with 5 ng/mL TGF-β in the presence of DMSO (0.1%) or SB203580 10μM. S10b) Confocal microscopy of stress fiber formation by TGF-β-induced EMT in AML12 cells. F-actin (red), nucleus pseudocolored green (SYTOX). Cells were stimulated as indicated in A).
HEP_22880_sm_SuppFig11a.TIF64KSupplementary S11. Densitometric quantification of ERK (S11a) and Akt phosphorylation (S11b) relative to GAPDH, from whole liver lysate, freshly isolated hepatocytes and hepatocytes cultured in CM or CS for the indicated times. S11c-d: Densitometric quantification of Akt (S11c) and ERK (S11d) from hepatocytes cultured in CM or CS and stimulated with 5 ng/mL TGF-β for 1h, on day 1 or day 2.
HEP_22880_sm_SuppFig11b.TIF65KSupplementary S11. Densitometric quantification of ERK (S11a) and Akt phosphorylation (S11b) relative to GAPDH, from whole liver lysate, freshly isolated hepatocytes and hepatocytes cultured in CM or CS for the indicated times. S11c-d: Densitometric quantification of Akt (S11c) and ERK (S11d) from hepatocytes cultured in CM or CS and stimulated with 5 ng/mL TGF-β for 1h, on day 1 or day 2.
HEP_22880_sm_SuppFig11c.TIF63KSupplementary S11. Densitometric quantification of ERK (S11a) and Akt phosphorylation (S11b) relative to GAPDH, from whole liver lysate, freshly isolated hepatocytes and hepatocytes cultured in CM or CS for the indicated times. S11c-d: Densitometric quantification of Akt (S11c) and ERK (S11d) from hepatocytes cultured in CM or CS and stimulated with 5 ng/mL TGF-β for 1h, on day 1 or day 2.
HEP_22880_sm_SuppFig11d.TIF61KSupplementary S11. Densitometric quantification of ERK (S11a) and Akt phosphorylation (S11b) relative to GAPDH, from whole liver lysate, freshly isolated hepatocytes and hepatocytes cultured in CM or CS for the indicated times. S11c-d: Densitometric quantification of Akt (S11c) and ERK (S11d) from hepatocytes cultured in CM or CS and stimulated with 5 ng/mL TGF-β for 1h, on day 1 or day 2.
HEP_22880_sm_SuppFig12a.TIF59KSupplementary S12. Densitometric quantification of Akt (S12a), ERK (S12b) and Smad2 (S12c) phosphorylation relative to GAPDH, from hepatocytes cultured in CM or CS treated with 5 ng/mL TGF-β for 48h, in the presence or absence of the indicated inhibitors. S12d: Western blot of GAPDH in comparison to other targets as seen in Fig. 6c. GAPDH band was cut however the equal load can be compared by the levels of p38.
HEP_22880_sm_SuppFig12b.TIF64KSupplementary S12. Densitometric quantification of Akt (S12a), ERK (S12b) and Smad2 (S12c) phosphorylation relative to GAPDH, from hepatocytes cultured in CM or CS treated with 5 ng/mL TGF-β for 48h, in the presence or absence of the indicated inhibitors. S12d: Western blot of GAPDH in comparison to other targets as seen in Fig. 6c. GAPDH band was cut however the equal load can be compared by the levels of p38.
HEP_22880_sm_SuppFig12c.TIF61KSupplementary S12. Densitometric quantification of Akt (S12a), ERK (S12b) and Smad2 (S12c) phosphorylation relative to GAPDH, from hepatocytes cultured in CM or CS treated with 5 ng/mL TGF-β for 48h, in the presence or absence of the indicated inhibitors. S12d: Western blot of GAPDH in comparison to other targets as seen in Fig. 6c. GAPDH band was cut however the equal load can be compared by the levels of p38.
HEP_22880_sm_SuppFig12d.TIF486KSupplementary S12. Densitometric quantification of Akt (S12a), ERK (S12b) and Smad2 (S12c) phosphorylation relative to GAPDH, from hepatocytes cultured in CM or CS treated with 5 ng/mL TGF-β for 48h, in the presence or absence of the indicated inhibitors. S12d: Western blot of GAPDH in comparison to other targets as seen in Fig. 6c. GAPDH band was cut however the equal load can be compared by the levels of p38.
HEP_22880_sm_SuppFig13a.TIF903KSupplementary S13: Re-epithelization of CM cultured hepatocytes. S13a) Hepatocytes cultured in CM were trypsinized and re-plated onto CM or in CS. 48h later, CMFDA was added. Fluorecent and phase contrast pictures were taken 30 min later. Active bile canaliculi secrete the fluorescent product of CMFDA hydrolysis. S13b) Real time PCR analysis of Claudin-1 in hepatocytes cultured in CM or CS for 3 days, and in cells trypsinized and re-plated into CM or CS for 48h.
HEP_22880_sm_SuppFig13b.TIF60KSupplementary S13: Re-epithelization of CM cultured hepatocytes. S13a) Hepatocytes cultured in CM were trypsinized and re-plated onto CM or in CS. 48h later, CMFDA was added. Fluorecent and phase contrast pictures were taken 30 min later. Active bile canaliculi secrete the fluorescent product of CMFDA hydrolysis. S13b) Real time PCR analysis of Claudin-1 in hepatocytes cultured in CM or CS for 3 days, and in cells trypsinized and re-plated into CM or CS for 48h.
HEP_22880_sm_Supptables.doc1095KSupplementary Table 1; Supplementary Table 2; Supplementary Table 3; Supplementary Table 4. Target genes with at leas 2 fold change of expression between day 2 and day 1 in CM culture.; Supplementary Table 5
HEP_22880_sm_Supptext.rtf51KSupplementary Text

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