Journal: Journal of Cellular and Molecular Medicine

Article Title: Activation of TGF-β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

doi: 10.1111/j.1582-4934.2008.00260.x

Figure Lengend Snippet: TGF-β-induced and spontaneous expression of CTGF in cultured rat PC ( A ) Western blot of CTGF of PC cultured for various times under serum-free conditions with or without addition of 5 ng/ml rh TGF-β1. Lysate was separated on 4–12% gel gradient in SDS (25μg protein/lane) under non-reducing conditions and probed for CTGF/CCN2 using a polyclonal anti CTGF-antibody. β-Actin always served as loading control. A representative blot of three independent experiments is shown. ( B ) CTGF/CCN2 reporter gene activation. PC were transfected simultaneously with 200 ng of reporter plasmid phCTGF-luciferase and 20 pg of promoterless Renilla -luciferase as internal control 1 hr after isolation and cultured under serum-free conditions with or without addition of 5ng/ml rh TGF-β1. Luciferase activity relative to Renilla-luciferase was measured 1 hr and 24 hrs after seeding. Mean values +/- SD of three independent experiments are given. ( C ) Western blot of CTGF of PC cultured for various times under serum-rich (10% FCS, top ) and serum-free ( bottom ) conditions, respectively. Two representative blots of four independent experiments are shown. ( D ) Western blot of CTGF of PC cultured under serum-free conditions with or without addition of actinomycin D and aphidicolin, respectively. Representative blots of three independent experiments are shown. ( E ) Immunoprecipitation of metabolically labeled CTGF. PC were cultured in serum-, cysteine- and methionine-free HepatoZYME-SFM for 24 hrs in the absence or presence of cycloheximide (5 μM) and pulse-labeled with the PRO-MIX L-[ 35 S] methionine/cysteine in vitro cell labeling kit each for 3 hrs before indicated time points. Culture medium was discarded, cells were washed, and cellular protein extracts were prepared, in which CTGF/CCN2 was immunoprecipitated with polyclonal anti-CTGF antibody and protein-G agarose beads. Immunocomplexes were resolved by RIPA+Complete, LDS and DTT. The radioactivity incorporated into CTGF was determined using a β-counter.

Article Snippet: Recombinant soluble TGF-β type II receptor (rsTβRII) was constructed as previously described [ ]; anti hTβRII (AF-241-NA), rhTGFβ-1 (240-B), rhLAP [TGF-β] (246-LP), recombinant latent TGF-β1, neutralizing anti TGF-β1 antibody (AF-101-NA) and mouse monoclonal anti-TGF-β1/-β2/-β3 (MAB1835) were from R&D Systems (Minneapolis, MN); calpain inhibitor III (#208722), calpain inhibitor IV (#208724), furin inhibitor II (#344931), human calpain I (#208713) and rat calpain II (#208718) were from Calbiochem (Darmstadt, Germany); Alk4/5 inhibitor (SB-431542) was from Tocris Bioscience (Ellisville, MO); actinomycin D (A1410), aphidicolin (A0781) and cycloheximide (#01810) were from Sigma Aldrich (St. Louis, MO); rabbit anti-Smad3 (ab28379) was from Abcam (Cambridge, UK); goat anti-CTGF/CCN2 (L-20, sc-14939) was from Santa Cruz (Santa Cruz, CA); rabbit anti-pSmad3 (Ser423/425)/pSmad1 (Ser463/465) (#9514), rabbit anti-pSmad2 (Ser465/467) (#3101) and rabbit anti-Smad2 (#3102) were from Cell Signaling/New England Biolabs (Ipswich, MA); and mouse anti-β-actin was from Cymbus Bioscience (Southampton, UK).

Techniques: Expressing, Cell Culture, Western Blot, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Isolation, Activity Assay, Immunoprecipitation, Metabolic Labelling, Labeling, In Vitro, Radioactivity

Journal: Journal of Cellular and Molecular Medicine

Article Title: Activation of TGF-β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

doi: 10.1111/j.1582-4934.2008.00260.x

Figure Lengend Snippet: Effect of external and internal inhibitors of TGF-β signalling on CTGF expression in PC cultured under TGF-β-free conditions ( A ) Western blot of CTGF of PC cultured for 24 hrs under completely serum-free conditions with or without addition of 5ng/ml rh TGF-β1. Some cultures received the Alk4/5 inhibitor (SB-431542, 5 μM), lysates were probed for CTGF/CCN2 and β-actin expression. A representative blot of four independent experiments is shown. ( B ) Immunoprecipitation of metabolically labeled CTGF. PC were cultured in serum-, cysteine- and methionine-free DMEM for 24 hrs in the absence or presence of Alk4/5 inhibitor (SB-431542, 5μM) and labeled with the PRO-MIX L-[ 35 S] methionine/cysteine in vitro cell labelling kit each for 3 hrs at indicated time points and processed as described in . ( C ) Western blot of CTGF of PC cultured for 24 hrs under complete serum-free conditions. Cultures received various inhibitors of extra- and intracellular TGF-β1 signalling like rsTβRII (1 μg/ml), neutralizing anti TGF-β1 antibody (4 μg/ml) and rhLAP [TGF-β] (1 μg/ml). Lysates were probed for CTGF/CCN2 and β-actin expression. A representative blot of three independent experiments is shown. ( D ) CTGF/CCN2 reporter gene activation. PC were prepared as described in , but received a blocking anti hTβRII antibody (10 μg/ml). They were cultured for 24 hrs under completely serum-free conditions. CTGF-luciferase activities are shown relative to Renilla luciferase activity. Mean values ± SD of four experiments are shown.

Article Snippet: Recombinant soluble TGF-β type II receptor (rsTβRII) was constructed as previously described [ ]; anti hTβRII (AF-241-NA), rhTGFβ-1 (240-B), rhLAP [TGF-β] (246-LP), recombinant latent TGF-β1, neutralizing anti TGF-β1 antibody (AF-101-NA) and mouse monoclonal anti-TGF-β1/-β2/-β3 (MAB1835) were from R&D Systems (Minneapolis, MN); calpain inhibitor III (#208722), calpain inhibitor IV (#208724), furin inhibitor II (#344931), human calpain I (#208713) and rat calpain II (#208718) were from Calbiochem (Darmstadt, Germany); Alk4/5 inhibitor (SB-431542) was from Tocris Bioscience (Ellisville, MO); actinomycin D (A1410), aphidicolin (A0781) and cycloheximide (#01810) were from Sigma Aldrich (St. Louis, MO); rabbit anti-Smad3 (ab28379) was from Abcam (Cambridge, UK); goat anti-CTGF/CCN2 (L-20, sc-14939) was from Santa Cruz (Santa Cruz, CA); rabbit anti-pSmad3 (Ser423/425)/pSmad1 (Ser463/465) (#9514), rabbit anti-pSmad2 (Ser465/467) (#3101) and rabbit anti-Smad2 (#3102) were from Cell Signaling/New England Biolabs (Ipswich, MA); and mouse anti-β-actin was from Cymbus Bioscience (Southampton, UK).

Techniques: Expressing, Cell Culture, Western Blot, Immunoprecipitation, Metabolic Labelling, Labeling, In Vitro, Activation Assay, Blocking Assay, Luciferase, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Activation of TGF-β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

doi: 10.1111/j.1582-4934.2008.00260.x

Figure Lengend Snippet: Effect of Alk4-/Alk5-inhibition on Smad-phosphorylation under TGF-β-free conditions ( A ) Western blot of p-Smad2, 1, 3 of PC cultured for 24 hrs under complete serum-free conditions and subjected to Alk4/5 Inhibitor (SB-431542, 5 μM), rsTβRII (1 μg/ml) and neutralizing anti TGF-β1 antibody (4 μg/ml), respectively. Lysates were probed for p-Smad2, p-Smad1, and p-Smad3, total Smad2, total Smad3, and β-actin, respectively. Representative blots of four independent experiments are shown. ( B ) Western blot of p-Smad3 (cross-reacting with p-Smad1) of PC cultured for 24 hrs under serum-free conditions with or without addition of rhTGF-β1 (5ng/ml). The blot is representative for three independent experiments.

Article Snippet: Recombinant soluble TGF-β type II receptor (rsTβRII) was constructed as previously described [ ]; anti hTβRII (AF-241-NA), rhTGFβ-1 (240-B), rhLAP [TGF-β] (246-LP), recombinant latent TGF-β1, neutralizing anti TGF-β1 antibody (AF-101-NA) and mouse monoclonal anti-TGF-β1/-β2/-β3 (MAB1835) were from R&D Systems (Minneapolis, MN); calpain inhibitor III (#208722), calpain inhibitor IV (#208724), furin inhibitor II (#344931), human calpain I (#208713) and rat calpain II (#208718) were from Calbiochem (Darmstadt, Germany); Alk4/5 inhibitor (SB-431542) was from Tocris Bioscience (Ellisville, MO); actinomycin D (A1410), aphidicolin (A0781) and cycloheximide (#01810) were from Sigma Aldrich (St. Louis, MO); rabbit anti-Smad3 (ab28379) was from Abcam (Cambridge, UK); goat anti-CTGF/CCN2 (L-20, sc-14939) was from Santa Cruz (Santa Cruz, CA); rabbit anti-pSmad3 (Ser423/425)/pSmad1 (Ser463/465) (#9514), rabbit anti-pSmad2 (Ser465/467) (#3101) and rabbit anti-Smad2 (#3102) were from Cell Signaling/New England Biolabs (Ipswich, MA); and mouse anti-β-actin was from Cymbus Bioscience (Southampton, UK).

Techniques: Inhibition, Western Blot, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Activation of TGF-β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

doi: 10.1111/j.1582-4934.2008.00260.x

Figure Lengend Snippet: Time course of APAAP immunostainings of TGF-β1, 2,3 in cultured PC ( A ) APAAP immunostainings of TGF-β in freshly isolated PC (0 hr) and in PC cultured for 24 hrs under serum-free conditions. A monoclonal antibody against the three isoforms of TGF-β was used. According to the manufacturer, the used mouse monoclonal anti-TGF-β1/-β2/-β3 antibody specifically detects the biologically active, mature peptide. Controls were performed with non-specific mouse immunoglobulin G (IgG) instead of TGF-β specific first antibody (Original magnification 40×). ( B ) APAAP immunostainings of TGF-β in PC cultured for 24 hrs under serum-free conditions but in the presence or absence of cycloheximide (5 μM) (Original magnification 40×).

Article Snippet: Recombinant soluble TGF-β type II receptor (rsTβRII) was constructed as previously described [ ]; anti hTβRII (AF-241-NA), rhTGFβ-1 (240-B), rhLAP [TGF-β] (246-LP), recombinant latent TGF-β1, neutralizing anti TGF-β1 antibody (AF-101-NA) and mouse monoclonal anti-TGF-β1/-β2/-β3 (MAB1835) were from R&D Systems (Minneapolis, MN); calpain inhibitor III (#208722), calpain inhibitor IV (#208724), furin inhibitor II (#344931), human calpain I (#208713) and rat calpain II (#208718) were from Calbiochem (Darmstadt, Germany); Alk4/5 inhibitor (SB-431542) was from Tocris Bioscience (Ellisville, MO); actinomycin D (A1410), aphidicolin (A0781) and cycloheximide (#01810) were from Sigma Aldrich (St. Louis, MO); rabbit anti-Smad3 (ab28379) was from Abcam (Cambridge, UK); goat anti-CTGF/CCN2 (L-20, sc-14939) was from Santa Cruz (Santa Cruz, CA); rabbit anti-pSmad3 (Ser423/425)/pSmad1 (Ser463/465) (#9514), rabbit anti-pSmad2 (Ser465/467) (#3101) and rabbit anti-Smad2 (#3102) were from Cell Signaling/New England Biolabs (Ipswich, MA); and mouse anti-β-actin was from Cymbus Bioscience (Southampton, UK).

Techniques: Cell Culture, Isolation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Activation of TGF-β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

doi: 10.1111/j.1582-4934.2008.00260.x

Figure Lengend Snippet: APAAP immunostainings of TGF-β1, 2, 3 in livers of CCl 4 - and D-galactosamine-injured rats Paraffin-embedded liver tissue of healthy rats and of rats treated intraperitoneally with CCl4 (1 week and 4 weeks) and D-galactosamine-HCl (D-GalN) (24 hrs), respectively were stained as described above (Original magnification 40β). Staining controls received a non-specific IgG instead of TGF-β specific first antibody.

Article Snippet: Recombinant soluble TGF-β type II receptor (rsTβRII) was constructed as previously described [ ]; anti hTβRII (AF-241-NA), rhTGFβ-1 (240-B), rhLAP [TGF-β] (246-LP), recombinant latent TGF-β1, neutralizing anti TGF-β1 antibody (AF-101-NA) and mouse monoclonal anti-TGF-β1/-β2/-β3 (MAB1835) were from R&D Systems (Minneapolis, MN); calpain inhibitor III (#208722), calpain inhibitor IV (#208724), furin inhibitor II (#344931), human calpain I (#208713) and rat calpain II (#208718) were from Calbiochem (Darmstadt, Germany); Alk4/5 inhibitor (SB-431542) was from Tocris Bioscience (Ellisville, MO); actinomycin D (A1410), aphidicolin (A0781) and cycloheximide (#01810) were from Sigma Aldrich (St. Louis, MO); rabbit anti-Smad3 (ab28379) was from Abcam (Cambridge, UK); goat anti-CTGF/CCN2 (L-20, sc-14939) was from Santa Cruz (Santa Cruz, CA); rabbit anti-pSmad3 (Ser423/425)/pSmad1 (Ser463/465) (#9514), rabbit anti-pSmad2 (Ser465/467) (#3101) and rabbit anti-Smad2 (#3102) were from Cell Signaling/New England Biolabs (Ipswich, MA); and mouse anti-β-actin was from Cymbus Bioscience (Southampton, UK).

Techniques: Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Activation of TGF-β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

doi: 10.1111/j.1582-4934.2008.00260.x

Figure Lengend Snippet: Modulation of TGF-β staining in cultured PC by calpain inhibitors and calpain APAAP immunostainings of TGF-β of PC were performed as described in . Cultured PC were treated with calpain 1 (0.5 U/ml), calpain 2 (0.5 U/ml), and cal-pain inhibitor III (20 μM), respectively for 24 hrs under serum-free conditions (Original magnification 40β).

Article Snippet: Recombinant soluble TGF-β type II receptor (rsTβRII) was constructed as previously described [ ]; anti hTβRII (AF-241-NA), rhTGFβ-1 (240-B), rhLAP [TGF-β] (246-LP), recombinant latent TGF-β1, neutralizing anti TGF-β1 antibody (AF-101-NA) and mouse monoclonal anti-TGF-β1/-β2/-β3 (MAB1835) were from R&D Systems (Minneapolis, MN); calpain inhibitor III (#208722), calpain inhibitor IV (#208724), furin inhibitor II (#344931), human calpain I (#208713) and rat calpain II (#208718) were from Calbiochem (Darmstadt, Germany); Alk4/5 inhibitor (SB-431542) was from Tocris Bioscience (Ellisville, MO); actinomycin D (A1410), aphidicolin (A0781) and cycloheximide (#01810) were from Sigma Aldrich (St. Louis, MO); rabbit anti-Smad3 (ab28379) was from Abcam (Cambridge, UK); goat anti-CTGF/CCN2 (L-20, sc-14939) was from Santa Cruz (Santa Cruz, CA); rabbit anti-pSmad3 (Ser423/425)/pSmad1 (Ser463/465) (#9514), rabbit anti-pSmad2 (Ser465/467) (#3101) and rabbit anti-Smad2 (#3102) were from Cell Signaling/New England Biolabs (Ipswich, MA); and mouse anti-β-actin was from Cymbus Bioscience (Southampton, UK).

Techniques: Staining, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Activation of TGF-β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

doi: 10.1111/j.1582-4934.2008.00260.x

Figure Lengend Snippet: Effects of calpain (inhibitors) on transcriptional activation of the CTGF-gene and subsequent CTGF protein expression ( A ) Western blot of PC cultured for 24 hrs under serum-free conditions in the presence or absence of calpain inhibitor III (10 μM), calpain inhibitor IV (1 μM) and furin inhibitor (20 μM), respectively. Analysis was as described in . A representative blot of four independent experiments is shown. ( B ) Western blot of PC cultured for 24 hrs under serum-free conditions in the presence or absence of calpain inhibitor III (10 μM), calpain inhibitor IV (1 μM) with or without addition of rh TGF-β1 (5 ng/ml). The blot is typical for three independent experiments. ( C ) Western blot of PC cultured for 24 hrs under serum-free conditions in the presence or absence of calpain inhibitor III (20 μM), calpain inhibitor IV (1 μM) with or without addition of rh TGF-β1 (5 ng/ml). One representative blot out of four independent experiments is shown. ( D ) CTGF/CCN2 reporter gene activation. Cultured PC were simultaneously transfected with hCTGF/CCN2-luciferase and promoterless Renilla -luciferase as internal control, subjected to calpain inhibitor III (20 μM) and cultured for 24 hrs under serum-free conditions. CTGF-luciferase activity relative to Renilla -luciferase is shown. Mean values ± SD of five experiments are shown. ( E ) RT-PCR for TGF-β1 and decorin in hepatic stellate cells (HSC) and PC cultured for 24 hrs. RT-PCR was performed using primers for TGF-β1 and decorin as described in Materials and Methods. Shown are the fragments for TGF-β1 and decorin after 32 cycles. The size of the amplified TGF-β1 fragment was 450 bp and the size of the amplified decorin fragment was 214kb. One representative result out of five is shown. ( F ) Activation of recombinant latent TGF-β (10 μg/ml) by calpain 1 and calpain 2 (each 0.5 U/ml) at 37°C. Thereafter, the concentration of immunologically detectable mature TGF-β was determined with an ELISA. Mean values ± SD of three experiments are given. ( G ) Activation of latent TGF-β in PC lysate at 37°C. Lysate of freshly isolated PC was kept untreated for 1 hr in ice or at 37°C. Other aliquots of the lysate received calpain 1 (0.5 U/ml) and calpain 2 (0.5 U/ml) prior to incubation. The concentration of mature TGF-β was quantified by an ELISA. Mean values ± SD of three experiments are given.

Article Snippet: Recombinant soluble TGF-β type II receptor (rsTβRII) was constructed as previously described [ ]; anti hTβRII (AF-241-NA), rhTGFβ-1 (240-B), rhLAP [TGF-β] (246-LP), recombinant latent TGF-β1, neutralizing anti TGF-β1 antibody (AF-101-NA) and mouse monoclonal anti-TGF-β1/-β2/-β3 (MAB1835) were from R&D Systems (Minneapolis, MN); calpain inhibitor III (#208722), calpain inhibitor IV (#208724), furin inhibitor II (#344931), human calpain I (#208713) and rat calpain II (#208718) were from Calbiochem (Darmstadt, Germany); Alk4/5 inhibitor (SB-431542) was from Tocris Bioscience (Ellisville, MO); actinomycin D (A1410), aphidicolin (A0781) and cycloheximide (#01810) were from Sigma Aldrich (St. Louis, MO); rabbit anti-Smad3 (ab28379) was from Abcam (Cambridge, UK); goat anti-CTGF/CCN2 (L-20, sc-14939) was from Santa Cruz (Santa Cruz, CA); rabbit anti-pSmad3 (Ser423/425)/pSmad1 (Ser463/465) (#9514), rabbit anti-pSmad2 (Ser465/467) (#3101) and rabbit anti-Smad2 (#3102) were from Cell Signaling/New England Biolabs (Ipswich, MA); and mouse anti-β-actin was from Cymbus Bioscience (Southampton, UK).

Techniques: Activation Assay, Expressing, Western Blot, Cell Culture, Transfection, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Incubation