Sh rna vectors

Unless otherwise specified all reagents are from Sigma-Aldrich. Opti-MEM, FluoroBrite™ DMEM, Lipofectamine® transfection reagent, DAPI, CellTrace™ CFSE Cell Proliferation Kit and CellTracker™ Orange Dye are from Invitrogen™ Life Technologies. siRNAs for Gal-1, RNase-free DNase set are from Qiagen. Sh-RNA vectors are purchased from OriGene. RNA Nano Chip kit is from Agilent. PrimeScript RT reagent kit, ProteaseMAX™ Surfactant, Sequencing Grade Modified Trypsin are from Promega. Polyvinylidene difluoride (PVDF) membrane is from Millipore. Transwells are purchased from Euroclone. Anti-Gal-1 antibody is from Cell Signaling, anti-β-actin and anti-Integrin-β1 antibodies are from Santa Cruz Biotechnology, anti-CD81 antibody is from BD Biosciences. The secondary antibodies enzyme horseradish peroxidase (HRP)–conjugated are from Santa Cruz Biotechnology. All materials for SDS-PAGE and Clarity™ Western ECL substrate are from Biorad. Culture-inserts (Dish 35 mm, high) are from ibidi®.

HEK-293T cells (ATCC) were cultured in DMEM-10% FBS medium. Human T-ALL cell lines Jurkat (ATCC), HSB-2 (ATCC), and Molt-4 (ATCC) were cultured in RPMI1640-10% FBS medium. USP7 shRNAs lentivirus was generated by co-transfecting 293T cells with shRNA vectors (OriGene or Sigma) (shRNA sequences listed in the Supplementary Table S5), and packaging plasmids. T-ALL cells were transduced with USP7 shRNA lentivirus and sorted for GFP positive cells five days after transduction or selected by puromycin. The knock-down of USP7 by shRNA was validated by real-time PCR and western blot. Genetically modified T-ALL cells were generated using CRISPR-Cas9 technology. Editing construct sequences and screening primers are listed in Supplementary Table S11.