Af1935

For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R&D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R&D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB). The secondary antibodies that were used were donkey anti-mouse Alexa488 (715-545-150, 1:400, Jackson ImmunoResearch), goat anti-mouse Alexa488 (115-545-146, 1:400, Jackson ImmunoResearch), goat anti-rat Alexa568 (A11077, 1:400, Invitrogen), donkey anti-rabbit Alexa647 (711-605-152, 1:400, Jackson ImmunoResearch). Blood vessels were visualized using Isolectin GS-IB₄ Alexa Fluor 568 conjugate (I21412, 1:250, Invitrogen). Images were collected on a confocal microscope (Zeiss LSM 510 unit mounted on an Axiovert 200 M inverted microscope) with 10x/0,3 EC Plan-NEOFLUAR Objective and/or with 20x/0,8 Plan-APOCHROMAT and on a Nikon AR1 confocal microscope with 40x/1,3 Plan-Fluor Objective. Image processing was performed using Zen 2011 and the NIH ImageJ software.

The cells were fixed with 1% formaldehyde at 37 °C for 10 min, and then rinsed twice with cold phosphate containing several protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, and 1 μg/mL pepsin A). Subsequently, the cells were incubated in pH 8.1 lysis buffer comprising of 1% SDS, 10 mM ethylene diamine tetraacetic acid (EDTA), and 50 mM Tris-HCl for 10 min, after which the genomic DNA was sliced using ultrasound. Following 10-min centrifugation of lysate at 13,000 r/min and 4 °C, the supernatant was diluted with a variety of reagents, including 0.01% SDS, 1% Triton X-100, 2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl (pH 8.1) and protease inhibitor. Anti-HIF1A (AF1935, R&D Systems, Minneapolis, MN) antibody was then supplemented to the lysate and cultured overnight at 4 °C. The following day, DNA fragments were recovered and PCR amplification was performed with the specific primers to detect the degree of enrichment of AQP4 promoter fragment binding to HIF1A.

Cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (ThermoFisher Scientific). For HIF western blot, cells were fractionated using RLN buffer (10 mM Tris–HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl₂, 0.5% NP-40, proteinase inhibitor cocktail) and the nuclear fraction was collected for analysis. Proteins were separated by SDS-polyacrylamide electrophoresis (SDS-PAGE), then transferred onto PVDF membranes (Biorad). Primary antibodies used in this study included those that recognized HIF-1α (R&D AF1935), HIF-2α (R&D AF2886; Cell Signaling #7096), β-actin (Thermo MA5-15739; Santa Cruz sc-1615), β-tubulin (Santa Cruz sc-5274), acetyl-histone H3 (Millipore 06-599), Lamin-B1 (Abcam ab16048), SREBP2 (R&D AF7119), hnRNPC (Santa Cruz sc-32308), TRIM28 (Abcam ab10483), RALY (Abcam ab170105), and Enterobacterio Phage MS2 coat protein (Millipore ABE76). For a complete list of antibodies used please see Supplementary Data 10.

Western blot analysis were performed by using (i) 40 μg of total protein extracts loaded on 10% polyacrylamide gels for TM9SF4, or (ii) 30 μg of nuclear protein extracts loaded on 7.5% polyacrylamide gels for HIF-1α, then transferred on membrane by using Transblot-Turbo transfer system, according manufacturer’s procedures (Bio-Rad, Hercules, CA, USA). Anti-TM9SF4 pAb (ab98879, Abcam, Cambridge, UK) and anti-HIF1α pAb (AF1935, R&D Systems, Minneapolis, MN, USA) were used according standard methods. Monoclonal antibodies anti-actin (Sigma-Aldrich, St Louis, MO, USA) and anti-nucleolin (Oncogene Research Products, Boston, MA, USA) were respectively used as internal control of total and nuclear proteins expression.