P erk1 2

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin, L-glutamine were obtained from GIBCO (Paisley, Scotland). DNase 1, gelatin, lypopolysaccharide (LPS), 1,10 phenanthroline (PA), poly-L-lysine (PLL), trypsin, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate, bovine serum albumine was provided by Sigma (St. Louis, MO, USA). Glial fibrillary acidic protein (GFAP) antibodies were purchased from Serotec (Oxford, UK). Standard proteins, G-250and R-250 coomassie brilliant blue were purchased from Bio-Rad (Hercules, CA, USA). Purified MMP-2 and MMP-9 were purchased from Alexis Biochemicals (San Diego, CA, USA). Primer pairs specific for MMP-2, MMP-9 and 18S were from Sigma Genosys (Cambridge, UK). RNeasy mini kit and QuantiTect Reverse Transcription were from Qiagen (Valencia, CA, USA). Antibodies against extracellular–regulated protein kinases (ERK) 1/2, and phosporilated ERK 1/2 (p-ERK 1/2) were from Santa Cruz Biotechnology (Santa Cruz, CA). Hybond-P PVDF membranes, enhanced chemiluminescence (ECL) Western Blotting Analysis System and anti-mouse-HRP secondary antibody were from GE Healthcare Life Sciences (Little Chalfont, Buckinghamshire, UK).

Equal amounts of total protein were separated by SDS–PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% albumin from bovine serum and then incubated overnight at 4 °C with the indicated primary antibodies, LRRN4 (Abcam, UK, ab133372), proliferating cell nuclear antigen (PCNA) (Calbiochem, Germany, 07-2162),CHK1 (Beyotime Biotechnology, China, AF18849), Phospho-Chk1 (p-Chk1, Cell Signaling Technology, USA, 2341S), Bcl-2 (Cell Signaling Technology, USA, 3498S), Bcl-xl (Abmart, China, Q07817), Caspase 3(Caspase 3/p17/p19 Monoclonal antibody, Proteintech, USA, 66470-2-Ig), Cleaved Caspase-3 (Cell Signaling Technology, USA, 9961S), Akt (Cell Signaling Technology, USA, 4691S), Phospho-Akt (p-Akt, Cell Signaling Technology, USA, 9271S), ERK 1/2 (Santa Cruz, UK, SC-514302), p-ERK 1/2(Santa Cruz, UK, SC-81492). After the appropriate secondary antibodies were added at room temperature, the proteins were detected with ECL reagent (SuperSignal West Pico Chemiluminescent Substrate, Pierce Biotechnology, USA) and visualized with the Electrophoresis Gel Imaging Analysis System (DNR Bio-Imaging Systems, Neve Yamin, Israel).

To detect the protein level of apoptosis-related molecules (Bax, Bcl-2, and cleaved caspase-3) and AQP-3, a western blot was performed according to the following steps. Total protein was isolated from NP cells using RIPA lysis buffer with PMSF and phosphatase inhibitor, and the concentration of the protein sample was measured with a BCA Protein Quantification kit. The same amount of proteins in each group was subject to SDS-PAGE and transferred to PVDF membranes. After being blocked with 5% skimmed milk at room temperature for 1 h, these PVDF membranes were incubated with primary antibodies (Bax: Proteintech, 60267-1; Bcl-2: Proteintech, 12789-1-AP; cleaved-caspase3: CST, 9661T; AQP-3: Abcam, ab125219; GAPDH: Proteintein, 60004-1-Ig; β-actin: Proteintein, 60008-1; ERK1/2: Santa Cruz, sc-292838; p-ERK1/2: Santa Cruz, sc-101761) at 4°C overnight with a dilution of 1 : 1000, then washed with TBST solution 3 times, and incubated with corresponding HRP-conjugated secondary antibodies (Beyotime, diluted 1 : 2000) at room temperature for 2 h. Then, protein bands were detected using the enhanced chemiluminescent system. GAPDH and β-actin were used as the internal reference.

N-Hydroxyphthalimide was purchased from Accela ChemBio Co., Ltd. (Shanghai, China). Propidium iodide (PI), RNase A and 4,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich. Antibodies of mTOR, Phospho-mTOR (Ser2448), Phospho-mTOR (Ser2481), S6K1, Phospho-S6K1 (Thr389), Phospho-S6 Ribosomal Protein (Ser235/236), 4E-BP1, Phospho-4E-BP1 (Ser65), Phospho-Akt (Ser473), Phospho-Akt (Thr308), Cleaved PARP, Cleaved Caspase 3, Caspase 9, cyclin B1, cdc2 were obtained from Cell Signaling Technology; antibodies of S6, Akt, P-ERK1/2, β-actin, Caspase 3, Caspase 8, Bcl-xL, survivin were from Santa Cruz; antibody of ERK was from Epitomics; antibody of eIF4E was obtained from BD Biosciences; Alexa Fluor® 647 donkey anti-mouse IgG antibody was purchased from Invitrogen; all the other secondary antibodies were from Sigma-Aldrich.

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.