Control shrna lentivirus

For all primary antibodies used in this study, see details in Table 1.
Wild-type APP770 plasmid and EGFP-tau (human tau441) plasmid were generous gifts from Prof. Angela Ho (Boston University, Boston, MA, USA) and Prof. Fei Liu (Jiangsu Key Laboratory of Neuroregeneration, China), separately. The APP^T668A was a mutant plasmid based on APP770 in which Thr742 (corresponding to the Thr668 site in APP695) was mutated to Ala. Wide-type PP2A plasmid was from Dr. Haendeler (University of Frankfurt, Germany). The surface-EGFP plasmid was constructed by inserting a Lyn before the EGFP sequence to enable EGFP to have the membrane-binding ability. pAAV-SYN-CIP2A-EGFP-3FLAG and control vector were from Obio Technology (Shanghai, China). CIP2A overexpression lentivirus, shRNA-CIP2A, and control shRNA lentivirus were constructed and packaged by Genechem (Shanghai, China). PCMV-u6-shRNA-CIP2A and control plasmids were constructed by Neuron Biotech (Shanghai, China); the target sequence of shRNA-CIP2A is 5’-GCACAATCTTTCTGTTCAA-3’.

The NLK shRNA and control shRNA lentiviruses were obtained from GeneChem (Shanghai, People’s Republic of China). The siRNA target sequences were: 5′-GAATATCCGCTAAGGATGC-3′ and 5′-CAGATCCAAGAGATGGAAA-3′. The stem-loop-stem oligonucleotide (shRNA) corresponding to each siRNA was inserted into the pGCSIL-green fluorescence protein (GFP) vector. Recombinant lentiviruses were produced by cotransfecting 293T cells with the lentivirus expression vector and packaging plasmids pHelper 1.0 and pHelper 2.0 (GeneChem) using Lipofectamine 2000 (Thermo Fisher Scientific). The supernatant was collected at 48 hours after transfection, and lentiviral particles were purified by ultracentrifugation (4,000× g) at 4°C for 10 minutes.
For cell infection, NCI-H446 cells were seeded in six-well plates at a density of 3×10⁴ cells/well and transduced with the constructed lentivirus-containing NLK shRNA (Lv-shNLK) or non-silencing shRNA (Lv-shCon) at a multiplicity of infection of 30. The lentiviral vectors expressed GFP, which allowed for measurement of infection efficiency in transduced cells.

The human NSCLC cell lines NCI-H1975, NCI-H1299, NCI-H1650, and A549 were purchased from the Shanghai Institute of Cell Biology Academia Sinica. All cancer cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, and 100 U/ml penicillin-streptomycin (Gibco-BRL; Thermo Fsiher Scientific, Inc.) at 37°C and 5% CO₂. The medium was changed after 24 h and replaced with fresh medium for transfection. Full-length NLK (Gene ID: 51701) was isolated from the human cDNA library. NLK-shRNA and control-shRNA lentiviruses were obtained from GeneChem Technologies. The shRNA target sequences were: 5′-GAATATCCGCTAAGGATGC-3′ and 5′-CAGATCCAAGAGATGGAAA-3′. A549 cells were infected with control-shRNA or NLK-shRNA lentiviruses according to the manufacturer's protocol.