S6639

Sorted cells were washed once with 1 mL PBS. Cells were fixed with 1 mL of MAA (methanol: acetic acid = 3:1) for 15 min on ice and spun down and re-suspended in 1 mL of MAA. This process is repeated for at least three times. Five million fixed cells were re-suspended in 1 mL of MAA. Cells were immobilized on the slide and denatured at 72°C for 3 min. Hybridization was performed in a dark humidity chamber for 3 days. Slides were washed with SSC buffer (S6639, Sigma), followed by staining with DAPI (P36935, Invitrogen). Slides were stored at −20°C or imaged immediately.

Tumor samples were fixed in 10% buffered formaldehyde and then decalcified in 4% EDTA 0.2% paraformaldehyde (PFA, pH 7.4) buffer for 4 weeks. After embedding in paraffin wax, 5 μm-thick sections were stained with Haematoxylin–Eosin–Safran (HES). Human nucleus detection was performed using a digoxin-labeled human locked nucleic acid Alu probe (Exiqon, Vedbaek, Denmark) as described previously [10] (link). Briefly, 70 nM Alu was hybridized on histological sections following DNA denaturation, in a buffer containing 4 X SSC (S6639, Sigma Aldrich, St. Quentin Fallavier, France), 50% deionized formamide, 1 X Denhardt's solution, 5% dextran sulfate and 100 µg/mL Salmon sperm DNA, for 19 h at 56 °C. Finally, the Alu probe was detected by peroxidase-based immunohisto-chemical procedure. For Tartrate-Resistant Acid Phosphatase (TRAP) detection slides were incubated 1 h in a 1 mg ml⁻¹ naphthol AS-TR phosphate, 60 mmol l⁻¹N,N-dimethylformamide, 100 mmol l⁻¹ sodium tartrate and 1 mg ml⁻¹ Fast Red TR salt solution (Sigma Aldrich, Saint Quentin Fallavier, France) and counterstained with haematoxylin. EGFP detection was observed on frozen sections with fluorescence microscopy directly or after immunostaining using mouse monoclonal to GFP primary antibody (Abcam, Paris, France) and Alexa Fluor 594 goat anti-mouse IgG (Life Technologies, Saint-Aubin, France).

The circ-FoxO3 conjugated with CY5 used for in situ hybridization was designed and synthesized by Sangon Biotech according to a previous study.²³ (link) Tissues or the BMECs (bEnd.3 or HBMEC) were washed twice with cold 1× DEPC PBS, and then were fixed with 4% PFA for 20 min. Permeability was performed for the tissues or BMECs with 0.25% Triton in PBS for 15 min. The samples were incubated in hybridization solution (Thermo Fisher Scientific, AM8670) containing 50 nmol/L CY5-labeled circ-FoxO3 probes at 55°C for 3 h after prehybridization in hybridization solution for 1 h at 37°C. Subsequently, the samples were washed with 2× SSC (Sigma-Aldrich, S6639) at 42°C and incubated in blocking buffer (0.5% BSA in PBST) before incubation with anti-CD31 antibody (Abcam, ab24590), PDGFR-β (CST, 3169), GFAP (CST, 3670), anti-mTOR (CST, 2983), anti-E2F1 (Abcam, ab112580), anti-SQSTM1/p62 (Abcam, ab56416), Alex Fluro 488-conjugated goat anti-mouse or rabbit IgG (Jackson Laboratory, 115-095-003; CST, 4412), and DAPI. Finally, the samples were captured using a Leica TCS SPII 5 confocal microscope (Leica, Solms, Germany). The sequences of probes are listed in Figure S1B.