Anti pai 1

Mice were sacrificed for histopathologic evaluation 14 weeks after irradiation. The skin and soft tissue of the irradiated leg was formalin-fixed, paraffin-embedded, cut into 4 μm sections, and stained with hematoxylin-eosin using standard procedures. To detect collagen accumulation in fibrotic tissues, Masson's trichrome staining was performed according to the manufacturer's protocol (Diagnostic BioSystems, Pleasanton, CA). For the immunohistochemical analysis, deparaffinized slides were incubated with anti-TGF-β1 (Cell Signaling), anti-PAI-1 (Santa Cruz Biotechnology), anti-SMA (abcam), anti-MMP2 (Santa Cruz Biotechnology), anti-MMP9 (Santa Cruz Biotechnology), anti-p300 (Santa Cruz Biotechnology), anti-CBP (Santa Cruz Biotechnology), and anti-acetylated p65 (Cell Signaling) primary antibodies, and subsequently with horseradish peroxidase-conjugated secondary antibodies. The DAB⁺ chromogen were used for signal detection (Dako Real Envision detection kit; Dako, Glostrup, Denmark). Representative images of the brown staining within fibrotic regions were captured and evaluated.

NIH-3T3 cells were pretreated with α-LA for 2 hours and then irradiated with 10 Gy using a linear accelerator (Varian, Palo Alto, CA). Cells were incubated for 12 hours and then analyzed by Western blot. The total cellular protein content was extracted with lysis buffer (50 mmol/L Tris-Cl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, and protease inhibitors). Proteins were separated on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and subjected to immunoblot analysis. Results were visualized by autoradiography. Protein expression was detected with anti-TGF-β1, anti-MMP9, anti-MMP2, anti-αSMA, and anti-PAI-1 (Santa Cruz Biotechnology, Cell Signaling Technology, Danvers, MA).

Western blot analyses were performed as previously described [21 (link)] with slight modifications. CnAOEC and CnAOSMC previously treated with 1 μg/ml of DiES for 24 h were lysed in ice-cold lysis buffer (20 mM Tris–HCl (pH 7.5), 140 mM NaCl, 10 mM ethylendiaminetetraacetic acid, 10% glycerol, 1% Igepal CA-630, aprotinin, pepstatin, and leupeptin at 1 μg/ml each, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate). Non-stimulated cells were used as controls under the same conditions. Protein samples (10 μg) were separated by SDS-PAGE under reducing conditions and blotted onto polyvinylidine difluoride membranes. Membranes were blocked before incubation with primary antibodies: anti-tPA, anti-uPA, anti-Annexin A2 and anti-PAI-1 (Santa Cruz Biotechnology Inc) according to the manufacturer’s recommendations. After incubation with HRP-conjugated secondary antibodies, bands were visualized by a luminol-based detection system with p-iodophenol enhancement. Anti-α-tubulin antibody (Oncogene Research Products) was used to confirm loading of comparable amount of protein in each lane. Protein expression was quantified by densitometry using Scion Image Software (Scion).

Cell lysates and kidney tissue homogenate were prepared, and Western blot analysis of protein expression was performed using routine procedures as described previously 26. The primary antibodies were used as follows: anti‐FHL2 (K0055‐3; Medical & Biological Laboratories, Japan), anti‐DDK (TA50011; OriGene), anti‐E‐cadherin (#610181; BD Transduction, San Jose, CA, USA), antifibronectin (F3648; Sigma‐Aldrich), anti‐α‐smooth muscle actin (α‐SMA) (A5228; Sigma‐Aldrich), anti‐β‐catenin (#610154; BD Transduction), anti‐dephosphorylated active‐β‐catenin (#05‐665; Millipore, Bellerica, MA, USA), anti‐phospho‐β‐catenin (Ser33/37/Thr41) (#9561; Cell Signaling, Boston, MA, USA), anti‐Snail (ab17732; Abcam, Cambridge, MA, USA), anti‐Twist (sc‐15393; Santa Cruz Biotechnology, Dallas, TX, USA), anti‐vimentin (#3932S; Cell Signaling), anti‐PAI‐1 (sc‐8979; Santa Cruz), anti‐MMP‐7 (GTX104658; GeneTex, Irvine, CA, USA), anti‐TBP (ab818; Abcam), anti‐GAPDH (sc‐25778; Santa Cruz Biotechnology) and anti‐α‐tubulin (sc‐53646; Santa Cruz Biotechnology).

Western blot analyses were conducted according to standard protocols. The following antibodies were used: anti-FAS, anti-Nrf2, anti-NQO1, anti-Srxn1, anti-PERK, anti-JNK, anti-TGFβ1, anti-Col1a1, anti-fibronectin, anti-α-SMA, and anti-PAI1 from Santa Cruz, (Santa Cruz, CA, USA); anti-eIF2α, anti-phospho-eIF2α (Ser51), anti-CHOP, anti-ERK, anti-phospho-ERK (Tyr204/Tyr187), anti-phospho-JNK (Thr183/Tyr185), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-Smad3, and anti-phospho-Smad3 (Ser423/425) from Cell Signaling Technology (Beverly, MA, USA); anti-phospho-PERK (Thr980) from Thermo Fisher Scientific; anti-phospho-Smad3 (Thr179) and anti-phospho-Smad3 (Ser208) from Invitrogen (Carlsbad, CA, USA); anti-TATA-binding protein (TBP) from Abcam (Cambridge, MA, USA); and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Merck Millipore. TBP and GAPDH were used as a loading control.

Moscatilin (Figure S3) was extracted and purified by one of our colleagues (Prof. Chien-Chih Chen) and the purity is more than 98%. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were acquired from Gibco (Grand Island, NY, USA). Anti-p-ERK1/2, -ERK1/2, -p-p70S6K, and anti-p-IκBα, were purchased from Cell Signaling Technologies (Boston, MA, USA). Anti-p-Akt, -Akt, -p-mTOR, -p-NF-κB/p65, and anti-GAPDH were obtained from Epitomics (Burlingame, CA, USA). Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and Anti-uPA, -uPAR, and anti-PAI-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).