SB 202190

CRC cells were incubated with PMPs as described above, and 50 × 10³ cells per ml were added to FITC-labelled gelatine-coated wells (0.1 mg/ml) and incubated for 24 h. The gelatinolytic activity of MMP-2 and MMP-9 was measured as green fluorescence derived from degraded gelatine (Additional file 2: Supplementary Methods). In another set of experiments, MMP-2 and MMP-9 activity was measured as CRC cell migration through 0.2% gelatin-coated filters in the Boyden chambers as described above (Additional file 2: Supplementary Methods). The gelatinolytic properties of CRC cell were blocked by the appropriate inhibitors of MMP-2 (ARP101) or MMP-9 (CTK8G1150) at a final concentration of 20 µM or by the specific inhibitor of p38MAPK (SB202190, Cell Signaling Technology) at a final concentration of 5 µM.

CTCs were cultured in RPMI medium supplemented with 20% foetal bovine serum, 10 µg Insulin, 20 mM HEPES, 10 µM Nicotinamide, 10 μM SB202190, 1.25 mM N-Acetyl-l-cysteine (Sigma-Aldrich, Ireland),10% (v/v) Noggin, 10 ng/mL FGF-10, 1 ng/mL FGF-2, 1X B27 Additive, 1:100 (v/v) Primocin (ThermoFisher Scientific, Dublin, Ireland), and 10 μM Y-27632 (STEMCELL Technology, Vancouver, Canada) and 100 µM CoCl₂ (Sigma-Aldrich, Ireland) using a 24 well low adherence plate (Sarstedt, Nümbrecht, Germany).

Cell culture plates were obtained from Greiner (Kremsmunster, Austria). μ-Slide 8 well plates were from Ibidi (Fitchburg, WI, USA). Reagents and biochemicals used during VSMC preparation were purchased from Duchefa Biochemie (Haarlem, The Netherlands) and Serva (Heidelberg, Germany). Collagenase type I was obtained from Worthington (Lakewood, NJ, USA). Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium and fetal bovine serum (FBS) were supplied by Biosera (Nuaille, France). The Opti-MEM medium used during transfection procedures was purchased from Gibco (Dublin, Ireland). Penicillin–Streptomycin (Sigma-Aldrich, Darmstadt, Germany) and GlutaMAX (Gibco) were used to supplement the cell culture medium. Molecular biology reagents, RevertAid Reverse Transcription Kit, GeneJet Gel Extraction Kit, and the restriction and ligase enzymes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). AngII and the inhibitors used in our study, namely: candesartan, SB202190, PD98059, and DPI were purchased from Sigma-Aldrich (St. Louis, MO, USA). JNK-IN-8 was purchased from Selleckchem (Houston, TX, USA). YM-254890 was obtained from Wako Chemicals (Neuss, Germany). TRV120023 (Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH) peptide was synthesized by Proteogenix (Schiltigheim, France) to more than 98% purity. For total RNA isolation, Qiagen’s (Hilden; Germany) RNeasy Plus Mini kit was used. Quantitative real-time PCR (qRT-PCR) reactions were prepared using the SYBR Green Kit (LightCycler 480 SYBR Green I Master) from Roche (Basel, Switzerland). Primer oligos were synthesized by Sigma-Aldrich. Paraformaldehyde was from Polysciences (Warrington, PA, USA), other reagents, and the primer monoclonal anti-Actin, α-Smooth Muscle, clone 1A4 antibody used in immunocytochemistry were supplied by Sigma-Aldrich. Fluorophore-conjugated secondary antibodies and Lipofectamine 2000 transfection reagent were from Invitrogen (Carlsbad, CA, USA). FuGENE 6 transfection reagent was supplied by Promega (Madison, WI, USA). NEB10 competent E. coli was obtained from BioLabs (Ipswich, MA, USA). Unless otherwise stated, all other chemicals and reagents were purchased from Sigma-Aldrich Merck (St. Louis, MO, USA). The mRFP-SAC1 DNA construct was a kind gift from Dr. Péter Várnai (Semmelweis University).