Anti mmp 9

Tissue protein was extracted with a protein extracting reagent (BioTeke Corporation, Beijing, China) according to the manufacturer's directions, while cell protein was extracted through RIPA buffer supplemented with 1% protease inhibitors (Roche, Basel, Switzerland). After centrifugation, the protein content was measured by a BCA Protein Assay Kit (Solarbio, Beijing, China). According to the standard procedures of western blot, total proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA), and then blocked with 5% skim milk in TBST. After being incubated with antibodies, the membranes were visualized with the ECL procedure (Millipore, USA) to get protein bands, which were analyzed by ImageJ software. The primary antibodies included anti-HMGN5, anti-Bcl-2, anti-Bax, anti-Cyclin D1, anti-p21, anti-MMP-2, anti-MMP-9, anti-AKT, anti-p-AKT, anti-ERK1/2, anti-p-ERK1/2 (Santa Cruz Biotechnology, Inc., USA), and anti-β-actin (WanleiBio, Shenyang, China). The secondary antibodies included HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (OriGene Technologies, USA).

Total protein from human PDAC cells was extracted by protein lysis buffer (Invitrogen) containing 2 μL protease inhibitor, according to the instruction. Protein concentration was determined by a BCA Protein assay kit (Millipore, Darmstadt, Germany). 30 mg of protein in each sample was separated by polyacrylamide gel electrophoresis via 10% separating gel and transferred to 0.22 µm PVDF membranes (Millipore). Then, membranes were blocked with 5% nonfat milk for 2 h at 37°C and followed incubated with anti-MMP-2 (no. sc-13594; 1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP-9 (no. sc-393859; 1 : 750; Santa Cruz Biotechnology), anti-N-cadherin (no. sc-59987; 1 : 1500; Santa Cruz Biotechnology), anti-E-cadherin (no. sc-8426; 1 : 2000; Santa Cruz Biotechnology), anti-Vimentin (no. sc-6260; 1 : 1000; Santa Cruz Biotechnology), anti-Snail (no. sc-271977; 1 : 500; Santa Cruz Biotechnology), and GAPDH (no. sc-47724; 1 : 5000; Santa Cruz Biotechnology) antibodies overnight at 4°C. Goat anti-mouse horseradish peroxidase-conjugated IgG (no. sc-2005; 1 : 2500; Santa Cruz Biotechnology) was used as the secondary antibody and incubated with the membranes for 1 h at 37°C. Finally, protein bands were observed using the enhanced chemiluminescence kit (Millipore) on Chemidoc XRS Gel Imaging System (Bio-Rad, Hercules, CA, USA).

Corilagin (CLG) was procured from Sigma-Aldrich (St. Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor^® 488 donkey anti-mouse IgG (H+L) antibody and Fluor^® 594 donkey anti-rabbit IgG (H+L) antibody was obtained from Life Technologies (Grand Island, NY, USA). Anti-MnSOD(sc-137254), anti-Fibronectin(sc-6952), anti-Vimentin(sc-6260), anti-E-cadherin(sc-8426), anti-N-cadherin(sc-271386), anti-Occludin(sc-5562), anti-Twist(sc-15393), anti-MMP-2(sc-53630), anti-MMP-9(sc-393859), anti-Wnt3a(sc-136163), anti-FZD-1(sc-398082), and anti-β-actin(sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail(3879S), anti-Axin-1 anti-Azin-1(3323S), anti-β-catenin(9562S), anti-p-GSK3β(9322S), and anti-GSK3β(9315S) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).

Ginkgolic acid C 17:1 (GAC 17:1) (95% purity) and ginkgolic acid C 15:1 (GAC 15:1) (98% purity), anti-STAT3, anti-SHP-1, anti-PTEN, anti-Bcl-2, anti-Bcl-xL, anti-survivin, anti-IAP-1, anti-COX-2, anti-MMP-9, anti-MMP-2, anti-caspase-3, anti-PARP, anti-β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris base, glycine, NaCl, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640, MEM, and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Annexin V was obtained from BD Biosciences (Palo Alto, CA, USA). Anti-p-STAT3 (Tyr705), anti-p-JAK1 (Tyr1022/1023), anti-JAK1, anti-p-src (Tyr416), anti-src, anti-cyclin D1, and anti-cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). pMXs-gw (#18656) and pMXs-STAT3C (#13373) were obtained from Addgene (Cambridge, MA, USA).

BT474 and SKBR3 cells were treated with plumbagin with the indicated concentrations and for the indicated time periods, after which cells were collected and lysed with the RIPA lysis buffer system (Santa Cruz, Heidelberg, Germany) for 30 min on ice. Lysates were centrifuged and supernatants were collected. Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with PBS buffer containing 5% non-fat dry milk for 1 h, then incubated overnight at 4°C with specific primary antibodies: anti-β-actin (1:1000) (Cell Signaling, Germany), anti-MMP-9 (1:200) (Santa Cruz, Heidelberg, Germany), anti-IKKα, anti-IKKα, anti-IκBα, anti-NF-κB (p65) (1:500) (Santa Cruz, Heidelberg, Germany), phospho-specific anti-NF-κB p65 (Ser536), (Cell Signaling, Germany), phospho-specific anti-IKKα (Ser176/180) (R&D Systems, USA). Membranes were next incubated with HRP-conjugated secondary antibodies (1:2000) (Cell Signaling Technology, Germany) at room temperature for 1 h. Proteins were visualized by chemiluminescence (ChemiDoc, Bio-Rad) with a HRP substrate (Pierce).

Immunohistochemistry was done using a single-staining procedure. Anti-WFCD2, anti-Ecadherin,anti-Vimentin monoclonal antibody (Cell Signaling Technology), anti-CD44,anti-MMP2,anti-MMP9,and anti-ICAM-1 rabbit polyclonal antibody (Santa Cruz Biotechnology), were applied to the slides at a dilution of 1:1,00 ~ 1:150 in blocking buffer overnight at 4 °C. The slides were then washed and stained by the avidin-biotin method. The slides were lightly counter stained with hematoxylin. Tumor cells were considered positive for the antigen if there was brown color staining. The intensity was scored as negative (0), weak (1), medium (2), and strong (3),and the proportion of staining was scored as 1 (≤10%), 2 (11–50%), 3 (51–75%), and 4 (>75%). An overall expression score was calculated by multiplying the scores for intensity and proportion, ranging from 0 to 12. For ICAM-1, at least 500 tumor cell for each xenograft sample (n = 5) were randomly selected and counted. The number of positive cell was counted and the positive index was calculated as follows: ICAM-1 index = (number of stained cells/total cell number) × 100%.