Af3420

For IP experiments the Chk1 (1:2, E250, Abcam) or GATA3 (1:2, 16E10A23, Biolegend) antibody was incubated with Red Protein G Affinity beads (Sigma-Aldrich) for 1 h at 4 °C on a shaker to allow the antibody to bind to the beads. Cell lysates were collected in RIPA buffer plus inhibitors (protease: complete mini Roche; 11836153001, and phosphatases: cocktail set II Calbiochem; 524625) and then incubated with the antibody-bead mix overnight at 4 °C on a shaker. The following morning, samples were centrifuged, and the supernatant was discarded leaving only the proteins able to bind to Chk1 or GATA3. SDS-polyacrylamide electrophoresis and western blotting of proteins of interest including, GATA3 (1:1000, 16E10A23, Biolegend), PGC1α (1:50, 3G6, Cell Signalling), Nrf1 (2 μg/ml, 492413, R&D Systems), Nrf2 (1 μg/ml, ab89443, Abcam), SOD3 (20 μg/ml, AF3420, R&D Systems), ATR (1:1000, E1S3S, Cell Signalling) and βactin (1:1000, 13E5, Cell Signalling) was then performed. Protein was detected via chemiluminescence by means of the ECL system (GE Healthcare, GERPN2106).

Microarrays were prepared from hCRC samples^{43 (link)} and IHC was performed in deparaffinized samples after antigen retrieval in citrate buffer (pH 6.0, 20 min) by incubation with goat anti-SOD3 (AF3420, R&D Systems), rabbit anti-HIF-2α (NB100-122, Novus Biologicals), or mouse anti-CD34 antibodies (QBEnd-10, M7165 Dako), followed by appropriate peroxidase-labeled secondary antibodies. The reaction was developed with AEC or diaminobenzidine (CD34) as chromogens, and hematoxylin counterstained. In all cases, sections from normal colon mucosa distant from the tumor site were used as controls. Staining was evaluated in a Leica DM500 optical microscope by a single pathologist (M.J.F.-A.) blinded to experimental data. The percentage of stained cells and staining intensity (scored as 1–3) were recorded for each gene, and the H-score was calculated as the product of these parameters^{63 (link)} for the epithelial tumor cell compartment and for tumor-associated stromal cells. In IF analyses, samples were incubated with all three antibodies, followed by appropriate secondary antibodies. Images were captured on an Olympus FluoView 1000 confocal microscope.