Eight- to ten-week-old and age-matched BALB/c nude mice have free access to water and food in a temperature-controlled room (23-25 °C) and relative humidity of 40-70%, the light/dark cycle is 12 hours, and there are 5 mice in each cage. Allow the animals to acclimate to the environment for at least 7 days before experiment. All animals were randomly and injected subcutaneously on both flanks with 1 × 106 Y79 and HXO-RB44 cells expressing the NC, gHELZ2, gHELZ2 + HELZ2. All animal use and experimental protocols were carried out in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines and approved by the Animal Care and Ethics Committee of Wuhan University of Medicine.
Y79 cells, HXO-RB44 cells and HEK293T cells were obtained from ATCC. All cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Biological Industries) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO2.
Y79 cells were transfected by Lipofectamine 2000. HEK293T cells were transfected by standard calcium phosphate precipitation.
The double-stranded oligonucleotides corresponding to the target sequences were cloned into the Lenti-CRISPR-V2 vector, and HEK293T cells were co-transfected with the packaging plasmid. Two days after transfection, the viruses were harvested, ultra-filtrated (0.45 mm filter, Millipore) and used to infect Y79 or HXO-RB44 cells in the presence of polybrene (8 mg/mL). Use puromycin (1 mg/mL) to select infected cells for at least 6 days.
Cell viability was assessed by the ability of cells to absorb Thiazole Blue Tetrazolium Bromide (MTT) (Bio-Tek, USA). The method is carried out in accordance with the manufacturer's instructions.
Immunoprecipitation and immunoblotting analysis
Cells were lysed in 1 ml NP-40 lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% Triton X-100, 10 μg/ml aprotinin, 10 μg/ ml leupeptin and 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation reaction, incubate 0.4 ml of lysate with 0.5-2 μg of the designated antibody or control IgG and 35 μL of 1:1 protein-G Sepharose (GE Healthcare) slurry at 4°C for 3 hours. Sepharose beads use 1 ml of lysis buffer containing 500 mM NaCl wash 3 times . The precipitates were fractionated by SDS-PAGE, and immunoblotting analysis was performed with the indicated antibodies. Antibodies used in this study were purchased from the following indicated companies: HA-Tag (#3724), Flag-Tag (#14793), c-Myc (#5605), or β-actin (#8457) from Cell Signaling Technology (MA, USA), or HELZ2 (#PA5-101743) from Invitrogen.
All data are expressed as the mean ± SE. Newman-Keuls tests were used for performing ad hoc comparisons when appropriate. Student’s t-tests and variance analyses were used to determine differences among groups. p < 0.05 was considered statistically significant.