Phenyl methyl sulfonyl fluoride (pmsf)

For protein extraction, adherent cells were washed with cold PBS and directly treated with cell lysis buffer (Cell Signaling, Danvers, MA, USA), freshly supplemented with 1 mM PMSF (Carl Roth, Karlsruhe, Germany) and a protease inhibitor cocktail (Cell Signaling, Danvers, MA, USA). Cells were scraped off the culture dish, incubated on ice, and centrifuged for 15 min. The Bradford assay, SDS-Page, semi dry transfer onto a polyvinylidene difluoride (PVDF) membrane, blocking, and incubation with the primary antibody were performed as described by Koch [16 (link)]. Primary antibodies against NOTCH1 (1:1000, NOTCH1 D1E11, Cell Signaling Technology, Inc., Danvers, MA, USA) or β-Tubulin antibody (1:1000, 2146, Cell Signaling Technology, Inc., Danvers, MA, USA) were used. After incubation, the membranes were washed in TBS-T and Anti-Rabbit IgG HRP-linked antibody (1:5000, Cell Signaling Technology, Inc., Danvers, MA, USA) was applied for another hour at 4 °C. The membranes were incubated with Thermo Scientific™ Pierce™ ECL Western Blotting Substrate (Fisher Scientific, Waltham, MA, USA) and chemiluminescence detection was performed with the ChemiDoc XRS+ Imager (Bio-Rad Laboratories GmbH, Munich, Germany). Analysis and editing were performed with Image Lab 6 (Bio-Rad Laboratories GmbH, Munich, Germany).

Cells were washed with ice-cold PBS and incubated in Triton buffer (0.5% Triton X-100, 50 mM MES, 25 mM EGTA, 5 mM MgCl₂) supplemented with 1 mM PMSF (Roth, Germany), Aprotinin, Pepstatin A (both Applichem, Germany), and Leupeptin (VWR, Germany) for 20 min on ice under continuous shaking. Thereafter, cell lysates were centrifuged at 13.000 rpm for 5 min, which leads to separation of the soluble cytosolic and insoluble cytoskeleton bound fraction. Subsequently, the pellets (insoluble fractions) were resuspended in SDS lysis buffer for Western blotting or with RIPA buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.1% SDS, 1% Nonidet P-40, 0.1 mM EDTA) for immunoprecipitation and followed by sonification. Protein concentrations were calculated as described above and equivalent amounts used for Western blotting or immunoprecipitation analyses.

The cells were lysed in lysis buffer (New England Biolabs, Frankfurt, Germany) supplemented with 1 mM PMSF (Roth, Karlsruhe, Germany). Equal amounts of protein (15 μg) were separated by SDS-PAGE and transferred to Immobilon PVDF membranes (Millipore, Schwalbach, Germany). The blocking of nonspecific binding sites was performed using 5% (w/v) non-fat dry milk in TBST. The membranes were then incubated with primary antibodies diluted in 5% non-fat dry milk/ TBST for 12-14 hours at 4 °C. HRP-conjugated immunoglobulins (diluted 1:5000 in 5% non-fat dry milk/TBST) were used as the secondary antibodies and incubated for 1 h at room temperature. Immunoreactivity was visualised using the Pierce ECL Western Blotting Substrate (Thermo Scientific) and exposure to high-performance chemiluminescence film (G&E Healthcare, Freiburg, Germany).

For protein expression analysis cells were lysed in 1× lysis buffer (New England Biolabs, Frankfurt, Germany) supplemented with 1 mM PMSF (Roth, Karlsruhe, Germany). Equal amounts of protein (15 μg) were separated by SDS-PAGE and transferred to Immobilion membranes (Millipore, Schwalbach, Germany). Blocking of unspecific binding sites was performed using 5% (w/v) non-fat dry milk in TBST. Membranes were incubated with primary antibodies diluted in TBST for 12–14 hours at 4°C. HRP-conjugated immunoglobulins (diluted 1:5000 in 5% non-fat dry milk/TBST) served as detection antibodies and were probed for 1 h at room temperature. Immunoreactivity was visualised by exposure to high-performance chemiluminescence film (G&E Healthcare, Freiburg, Germany). We used primary antibodies against p-Akt Ser 473 (rabbit, clone D9E) 1:500 (Cell Signaling), p-Erk1/2 Thr 202/Tyr 204 (rabbit) 1:1000 (Cell Signaling), Survivin (rabbit) 1:1000 (Cell Signaling), Aurora-Kinase A (rabbit) 1:500, Aurora-Kinase B (rabbit) 1:500 and Tubulin (rabbit) 1:5000.

A total of 60,000 cells per well were seeded in 2.5 mL culture media in six well plates 1 day before the transfection. Triplicates were made. For the transfection 1.5 mL of Opti-MEM and 15 μL of Lipofectamine RNAiMax (Life Technologies), were mixed and incubated for 5 min at room temperature. After 5 min 400 ng of esiRNA were incubated for 20 min. Then 500 μL of the mix were added dropwise to each well. After 6 days cells were counted. Additionally, the triplicates were pulled, centrifuged 5 min, 4°C, 13,500 rpm, washed with PBS and resuspended with lysis buffer (1 mL of 10x lysis buffer (Cell Signaling), one tablet of Protease Inhibitor (Roche), 200 μL of phosphatase inhibitor cocktail III (Merck), 5 μL of 200 mM PMSF (Carl Roth), filled up to 10 mL). After 10 min incubation on ice, samples were centrifuged 10 min, 4°C, 13,500 rpm, and supernatants were transferred to new tubes. Protein determination was assessed with BCA Kit (Pierce). Western blot was performed according with standard procedures. The antibodies used were: RASSF8 (4B1) mouse monoclonal (Santa Cruz, 1:250), goat anti mouse HRP (Millipore, 1:3,000), and ß-actin HRP conjugated (Santa Cruz, 1:3,000).

TNF was purchased from Sigma-Aldrich (Darmstadt, Germany) and PMSF (a protease inhibitor applied for validation experiments) from Roth (Karlsruhe, Germany). Inhibitor experiments were performed using the GSK3α/β inhibitor Kenpaullone (Sigma-Aldrich). For Western blotting, antibodies specific for CD44 (156-3C11), p-C/EBPβ (Thr235), GSK3β (27C10), IDO (D5J4E), MARCKS (D88D11), NFKB1-p105/p50, NFKB2-p100/p52, p65 (L8F6), RELB (C1E4), VIM (5G3F10), p-VIM (Ser56; all from Cell Signalling, Danvers, MA, USA), p-CD44 (Ser706; Santa Cruz Biotechnology, Santa Cruz, CA, USA), C/EBPβ (E299; Abcam, Cambridge, UK), KYNU (Biozol, Eching, Germany), p-MARCKS (Ser159), p-RELB (Ser573; Thermo Fisher), p-GSK3α/β (Tyr279/Tyr216), actin (11C), GAPDH, and TBP (Sigma-Aldrich) were used. Horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signalling or Vector Laboratories (Burlingame, CA, USA). All media and reagents were of the best available grade and routinely tested for endotoxins with the Limulus Amoebocyte Lysate assay (Lonza, Basel, Switzerland).