Mmp 9

DMEM, fetal bovine serum (FBS), and phosphate buffered saline (PBS) were purchased from Gibco (Grand Island, NY). Primary antibodies for SCAI, MMP-9, E-cadherin, and β-actin were obtained from Santa Cruz Biotechnology Co., Ltd. (Santa Cruz, CA, U.S.).

We used antibodies against Src, p-Src, Snail, TGFβ, Lrp5, Runx2 (Cell Signaling, Danvers, MA, USA), DRD₁, DRD₂ (Abcam, Cambridge, MA, USA), MMP9 (Santa Cruz Biotechnology, Dallas, Texas, USA), TH (Novus Biologicals, Centennial, CO, USA), CCN4 (R&D Systems, Minneapolis, MN, USA), and β-actin (Sigma). Protein levels were assayed using a SuperSignal West Femto maximum sensitivity substrate (Thermo-Fisher). The level of dopamine in the serum was determined using an ELISA kit (MyBioSource, San Diego, CA, USA). We also employed a mouse XL cytokine array (R&D Systems, Minneapolis, MN, USA) and determined the expression of 111 cytokines and chemokines in mouse serum samples.

Cells were lyzed with RIPA buffer and total 30 μg of protein was loaded on 6–12% SDS-PAGE. After transferring to PVDF membranes, each membrane was blotted with the appropriate antibodies. Anti-PARP, -p-EGFR, -EGFR, -p-STAT3, -STAT3, -p-JAK1, -p-JAK2, -p-AKT, and -AKT antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p-SRC, -SRC, -p-ERK1/2, -ERK1/2, -VEGF, -Cyclin D, -MMP-9, -Survivin, and -Tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immunofluorescence assays for p-STAT3 nuclear translocation in MDA-MB-231 cells were done with anti-p-STAT3 antibody and anti-Alexa Fluor-488 antibody (Invitrogen, Eugene, OR, USA). For the counter staining, TOPRO-3 (Invitrogen, Eugene, OR, USA) was used to stain the nucleus. Images were obtained with Olympus FV10i Self-Contained Confocal Laser System.

Immunoblot analyses were performed as described previously [10 (link)]. Antibodies specific for MMP9 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc-6841), Bid (Cell Signaling Technology, Boston, MA, USA, #2002), cleaved-PARP (Cell Signaling, #5625), cleaved-caspase 3 (Cell Signaling, #9664), and GAPDH (Santa Cruz; sc-32233) were used. Immunoreactive proteins were detected by chemiluminescence using X-ray films.

The immunoblot procedure was performed as described previously [14 (link)]. Antibodies specific for cyclin E, CDK2, CDK4, fascin, MMP-9, vimentin, and Ub (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), cyclin D, (Abcam, San Francisco, CA, USA), N-cadherin (Thermo Fisher Scientific Inc.), and GAPDH (Merck Millipore, Billerica, MA, USA) were employed. Band intensities were calculated using Image Gauge software (Fujifilm, Tokyo, Japan). The intensities of target gene expression were normalized to GAPDH signals for lysate and fascin signals for supernatant proteins.

Immunohistochemistry was performed to test target proteins expression in samples from clinical patients and tumour xenographs by standard protocols. Briefly, paraffin sections (4 μm) were deparaffinized and rehydrated, and heat-induced antigen retrieval was conducted in sodium citrate buffer (10 mM, pH 6.0). Endogenous peroxidases were blocked by incubation in 0.3% H₂O₂. The sections were then incubated with primary antibodies against FMNL3 (Cat. #HPA023201; Sigma, Germany), E-cadherin (Cat. #3195 S; Cell Signaling, MA, USA), Vimentin (Cat. #5741 S; Cell Signaling) and MMP-9 (Cat. #sc-21733; Santa Cruz) at 4 °C overnight. Non-immune IgG was used as a negative control. Antigenic sites were visualized using a SP9000 and DAB kits (ZSGB-BIO, Beijing, China). The immunoreactive scores (IRS) of FMNL3, E-cadherin, Vimentin and MMP-9 were calculated as follows: 0, negative; 1, weak; 2, moderate; or 3, strong. The percentage of positive cells was scored as follows: 1, 0–9% positive cells; 2, 10–50% positive cells; and 3, >50% positive cells. The two scores were multiplied together and samples with a total IRS of 0, 1–3, 4–6 and 7–9 were considered as (−), (+), (++) and (+++), respectively.