Primers used for RT-PCR analysis.

<p>F: forward primer, R: reverse primer.</p

Antifibrotic effect of FK506 in alcohol model of hepatic fibrosis.

<p>A. Masson's trichrome staining of the liver sections from rats treated for 4 weeks as indicated. Two livers of each treatment group are shown at 100× magnification. B. 500× magnification of the squared sections in A. C. Percentage of liver fibrosis was determined from the Masson's trichrome staining using ImageJ software and plotted as percent fibrotic area vs. total liver area. Data represents average and±1 SEM from 8 rats, *** represents significance at p<0.01. D. and E. Aminotransferases in plasma of the experimental animals. The activity is presented as average and±1 SEM of 8 animals analyzed in duplicate.</p

FK506 reduces collagen synthesis by precision cut liver slices.

<p>The 350 µm thick rat liver slices were analyzed immediately after the preparation (day 0, lanes 1–3) or after culturing for 3 days without FK506 (day 3, lanes 4–6) or with two different concentrations of FK506 (day 3, lanes 7–12). Expression of collagen α1(I) polypeptide, αSMA and actin, as a loading control, was analyzed by western blot. α1(I) collagen polypeptide is resolved as processed, mature, polypeptide of 120 kDa (mature collagen) and as freshly synthesized unprocessed α1(I) pro-peptide of 180 kDa (pro-collagen). A nonspecific band is indicated by asterisk.</p

Interaction of LARP6 with FK506 binding protein 3 (FKBP3).

<p>A. Schematic representation of LARP6 constructs used in immunoprecipitations (IP) with amino-acid numbering on the top. All constructs contained HA tag at the N-terminus. Full size LARP6 (FS) has the domains indicated: N-TER, N-terminal domain, La, La homology domain, RRM, RNA recognition motif, C-TER, C-terminal domain. Binding of the constructs to 5′ SL RNA is indicated to the right. B. IP of endogenous FKBP3 with LARP6 constructs. Upper panel: after expression of HA-tagged LARP6 constructs in HEK293 cells, IP was performed with anti-HA antibody and Western blot with anti-FKBP3 antibody. Bottom panel: Expression of the proteins in the input material analyzed by Western blot. C. IP of endogenous LARP6 with endogenous FKBP3 in cells treated with FK506. Top panel: HLFs were treated with the indicated concentrations of FK506. The IP was done using anti-FKBP3 antibody (lanes 1 and 2) or anti-fibronectin antibody (FIB, lanes 3 and 4), as control, and Western blot probed with anti-LARP6 antibody. Bottom panel: expression of proteins in the input material.</p

Abbreviations.

<p>Abbreviations.</p

Expression of LPS-BP mRNA and CD64 mRNA in the livers of experimental animals.

<p>A. Total RNA from the livers of treated animals was analyzed by RT-PCR for expression of lipopolysaccharide binding protein mRNA (LPS-BP) and CD64 mRNA. Data from 4 animals of each group are shown. Loading control, actin (ACT). B. Quantification of expression of LPS-BP mRNA after normalization to actin mRNA expression. The data from all 8 animals were used and presented as average and±1 SEM. * represents significance at p<0.05. C. Quantification of expression of CD64 mRNA after normalization to actin mRNA. *** represents significance at p<0.01 and * represents significance at p<0.05.</p

FK506 inhibits secretion of collagen polypeptides into cellular medium.

<p>A. Western blot analysis of collagen α1(I) and α2(I) polypetides in cellular extracts and in the medium of human lung fibroblasts (HLF) treated with the indicated concentrations of FK506. Top panel: western blot of cellular collagen. Loading control: tubulin. Middle panel: Western blot of collagen secreted into the cellular medium. Loading control, fibronectin (FIB). Lower panel: RT-PCR analysis of the collagen mRNAs level in HLFs. Actin mRNA (ACT) is shown as loading control. B. Western blot analysis of collagen α1(I) polypeptide in cellular extracts and in the medium of primary activated rat HSCs (rHSCs) treated with the indicated concentrations of FK506. Top panel: Collagen α1(I) polypeptide in cellular extracts. α-smooth musle actin (αSMA) was analyzed as a marker of HSCs activation and tubulin as loading control. Middle panel: secretion of collagen α1(I) polypeptide into the medium of rHSCs. Lower panel: RT-PCR analysis of the collagen mRNAs level in rHSCs. C. Same analysis as in B, except activated human hepatic stellate cell line (hHSC) was used.</p

FK506 inhibits association of collagen mRNAs with FKBP3.

<p>A. Expression of proteins in the input material used for mRNA pull downs analyzed by Western blot. B. Top panel: pull down of collagen mRNAs with FKBP3. HLFs treated with 2 µM FK506 (lanes 1 and 2) or untreated HLFs (lanes 3 and 4) were used for IP with anti-FKBP3 antibody (lanes 2 and 4) or anti-fibronectin antibody (lanes 1 and 3). The IP material was analyzed for collagen α1(I) (COL1A1), collagen α2(I) (COL1A2) and actin (ACT) mRNA by semi-quantitative RT-PCR. Bottom panel: analysis of the mRNAs in the input material. C. Quantitative RT-PCR (qRT-PCR) analysis of the collagen mRNAs in the IP from A. The experiments were done in duplicates and plotted as fold change over the negative control, actin mRNA. The error bars represent average with±1 SEM. *** represents significance with p<0.01.</p

FK506 reduces expression of type I collagen and αSMA in hepatic fibrosis.

<p>A. RT-PCR analysis of collagen α1(I) mRNA (COL1A1), collagen α2(I) mRNA (COL1A2), αSMA mRNA and actin (ACT) mRNA in total liver RNA from the experimental animals. The results from 4 animals in each group are shown. B. and C. Quantification of expression of collagen α1(I) and α2(I) mRNA after normalization to actin mRNA expression. The data from all 8 animals were used and presented as average and±1 SEM. *** represents significance at p<0.01. D. Western blot analysis of total proteins from the livers of experimental animals. Collagen α1(I) polypeptide (COL α1(I)), αSMA and actin (ACT), as a loading control, from 4 animals of each group is shown. E. Quantification of expression of collagen α1(I) polypeptide after normalization to actin expression. The data from all 8 animals were used and presented as average and±1 SEM. *** represents significance at p<0.01. F. Quantification of αSMA expression after normalization to actin expression.</p

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

<div><p>Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties <em>in vitro</em> and <em>in vivo</em> and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited <em>in vitro</em> activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis.</p> </div