5 fluorouracil 5 fu

ABT-869, 5-Fluorouracil (5-Fu) and cisplatin were purchased from Selleck Chemicals (Houston, TX). Recombinant human VEGF (VEGF₁₆₅) was a gift from the Experimental Branch of the National Institutes of Health (Bethesda, MD). Recombinant human epidermal growth factor (EGF) and insulin-like growth factor (IGF) were purchased from R&D Systems (Minneapolis, MN). Growth factor-reduced Matrigel was obtained from BD Biosciences (San Diego, CA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): VEGFR2, EGFR, IGF1R, AKT, mTOR, p70 S6K Kinase (S6K), eukaryotic initiation factor 4e (4EBP-1), poly (ADP-ribose) polymerase (PARP), Bcl-2, Bcl-xL, CyclinD1, phospho-specific anti-EGFR (Tyr¹⁰⁶⁸), anti-IGF1R (Tyr¹³¹⁶), anti-VEGFR2 (Tyr¹¹⁷⁵), anti-AKT (Ser⁴⁷³), anti-mTOR (Ser²⁴⁴⁸), anti-S6K (Ser^235/236) and anti-4EBP-1 (Thr^37/46). Anti-CD31 antibody was obtained from Epitomics (Burlingame, CA).

Determination the IC50 of BCA cells in response to chemotherapeutic drugs was analyzed by MTS assay. The experiment started from 5 × 10³ of BCA cells in 100 μl medium that were seeded in 96-well plates for 24 h, then media was changed with different concentrations of new chemotherapeutic drugs [doxorubicin (S1208), paclitaxel (S1150), cisplatin (S1166), 5-fluorouracil (5-FU) (S1209) and gemcitabine (S1714) that were purchased from Selleck Chemicals, Houston, TX, USA] with or without rPN and anti-PN peptide for a further 48 h. After that, cell viability was determined by the MTS assay. Combination index (CI) was calculated by IC50 of combination of drug and PN or anti-PN peptide divided by IC50 of chemotherapeutic drug alone.

Human embryonic kidney (HEK) 293T cells, normal human colon epithelial NCM460 cells and CRC cells including HCT8, RKO, CACO2, SW620, SW480, HCT116 and DLD1 were purchased from the Cell Bank of the Chinese Academy of Sciences. These cells were cultured in RPMI 1640 (Hyclone, China) supplemented with 10% Fetal Bovine Serum (FBS, Gibco-BRL, Invitrogen). They were cultured in a humidified 5% CO₂ incubator at 37 °C, and all the experiments were carried out during their logarithmic growth phase. Chemotherapeutic agents such as 5-fluorouracil (5-FU, Selleck, China) and oxaliplatin (L-OHP, MCE, China) was administrated to CRC cells at a final concentration of 800 μg/ml and 6 mg/ml, respectively, to investigate the chemosensitivity of CRC cells.
The shRNA and siRNA targeting HTRA1 and SLC7A11 were synthesized by Tsingke Biotech (Beijing, China). The wild-type HTRA1 overexpression vector with FLAG tag (pEnter-HTRA1) was purchased from Generay Biotech (Shanghai, China). All plasmids, siRNAs and shRNAs were transfected into CRC cells using Lipofectamine 3000 (Invitrogen, USA). HTRA1 stable overexpressing CRC cells were obtained by antibiotic screening after transfection. All siRNA and shRNA sequences are listed in Supplementary Table S1.

The human GBM cell line U87 was purchased from the Chinese Academy of Science (Shanghai, China) and was verified using short tandem repeat assays by GENEWIZ (Suzhou, China). All cells were grown in DMEM (Hyclon, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Beit-Haemek, Israel), 1% penicillin and streptomycin (P/S; Hyclon). To initiate stem-like induction, the cells derived from U87 xenografts were cultured in a defined serum-free neural stem cell (NSC) medium containing 20 ng/ml of basic fibroblast growth factor (bFGF, Peprotech, Rocky Hill, NJ, USA), 20 ng/ml of epidermal growth factor (EGF, Peprotech), N2 and B27 (Invitrogen). TMZ, Doxorubicin (DOX), Etoposide (Eto), Cis-platinum (CIS), and 5-Fluorouracil (5-Fu) were acquired from Selleck Chemicals.

A375 and A375R cells were seeded at a density of 3 × 10³ and 4.5 × 10³ cells per well, respectively, in 96-well plates. Twenty-four hours later, Vem at concentrations from 0 to 2.5 μM for A375 and 0–80 μM for A375R, cisplatin (Selleckchem, United States) with the concentration from 0 to 40 μM, 5-fluorouracil (5-Fu) (Selleckchem, United States) with the concentration from 0 to 50 mg/ml, bufalin (TargetMol, United States) with the concentration from 0 to 200 nM, IMD-0354 (Selleckchem, United States) with the concentration from 0 to 10 μM, and NCTD (TargetMol, United States) at concentrations from 0 to 400 μM were added to the medium for 48 h of treatment. Briefly, cell viability was analyzed by Cytation 5 (BioTek, United States) at 450 nm after using the Cell counting kit-8 (HY-K0301, MedChemExpress) for 2 h of incubation, and the 50% inhibitory concentrations (IC₅₀) values were calculated.