Control shrna

To silence SIRT1, HepG2 cells were transfected with siRNA against human SIRT1 (33–100 nM) using Lipofectamine 2000 according to the manufacturer’s guidelines. The siRNA sequence was synthesized by Sangon Biotech (Shanghai, China) (SIRT1 siRNA: sense, 5′-UAGGUGCCCAGCUGAUGAAUU-3′, anti-sense, 5′-UUCAUCAGCUGGGCACCUAUU-3′; negative control siRNA: sense, 5′-UUCUCCGAACGUGUCACGUUU-3′, anti-sense, 5′-ACGUGACACGUUCGGAGAAUU-3′).The shRNA sequence (SIRT1 shRNA, 5′-GATCCTAGGTGCCCAGCTGATGAGTTCAAGAGACTCATCAGCTGGGCACCTATTTTTG-3′ or control shRNA, 5′-GATCCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3′) was synthesized by Sangon Biotech and inserted into the pGreenPuro vector (SBI). Recombinant lentiviruses encoding mouse SIRT1 shRNA (LV-shSIRT1) or negative control shRNA (LV-shCtrl) were treated as described previously34 (link).

shRNA sequences were inserted into a PSIREN-RetroQ Vector (Clontech Laboratories, Inc., CA, USA), and then, HEK293T cells were co-transfected with the construct and packaging plasmids (pGag-Pol and pVSV-g). At 48 h posttransfection, the supernatants were collected and filtered using a 0.45-μm cellulose acetate filter. Further, the JURKAT and MOLT-4 cells were incubated in culture medium with supernatants containing virus particles and supplemented with 8 μg/mL polybrene (Sigma-Aldrich) for 8 h, followed by replacement with fresh medium. At 48 h postinfection, puromycin (2 μg/mL; Calbiochem, Merck KGaA, Darmstadt, Germany) was added to screen the cells. Control shRNA was synthesized by Sangon Biotech (Shanghai, China), and the following USP7 shRNA-1 and USP7 shRNA-2 sequences were used: USP7 shRNA-1, 5′-TGCGAAATCTGCCATGGAA-3′; USP7 shRNA-2, 5′-CTCAGAACCCTGTGATCAA-3′.