Lentiviral particles

BALB/c nude mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (male, 6 weeks old, 18-20 g). Animal experiments were approved by the Institute of Basic Medicine, Shandong First Medical University & Shandong Academy of Medical Sciences and were performed according to the established guidelines. Mice were fed under the condition of specific pathogen-free. shERCC6L and NC were used to construct lentiviral particles (GenePharma), and the lentivirus were infection to establish stable transfected cells. Mice were randomly grouped two groups (with 7 mice each group): negative control group (NC group, injected with SMMC7721 transfected with NC) and shERCC6L group (injected with SMMC7721 transfected with shERCC6L). A total of 1 × 10⁷ cells were suspended in 100 μL serum-free DMEM and injected subcutaneously into the right back of mice. After injection, the long diameter (L), width diameter (W) of tumors were measured every 5 days, and the volumes of tumor were calculated as following Volume (V) = (W² × L)/2. Mice were anaesthetized by intraperioneal injection of pentobarbital sodium overdose (100 mg/kg) on the 30th day. Euthanasia was considered to be successful if there was cardiac arrest and no spontaneous breath for 3 min, and then tumors were taken out and weighed.

Lentivirus expressing scramble or ALKBH5 shRNAs was purchased from GenePharma Company. In the case of knockdown experiments, cells were infected these lentiviral particles and selected with 3 μg/ml puromycin. In the case of overexpression experiments, cells were infected with lentiviral particles expressing empty vector control or ALKBH5 (GenePharma Company) and selected with 3 μg/ml puromycin.

Short hairpin RNA (shRNA) sequences were obtained from Public TRC Portal (Broad Institute, Cambridge, MA, USA) and lentiviruses expressing the shRNA sequences were synthesized by Shanghai GenePharma Co., Ltd., (Shanghai, China). Polybrene and puromyocin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Transfection of the HepG2 cells with lentiviral particles (Shanghai GenePharma Co., Ltd.)was conducted as described previously (23 (link)). Titration of the lentiviruses was performed (not shown), and the lowest functional quantities of the virus (MOI=5) and Polybrene were subsequently applied in the LOF experiments. The shRNA target sequences for PKR were as follows: shPKR-1, 5′-GCTGAACTTCTTCATGTATGT-3′ and shPKR-2, 5′-GAGGCGAGAAACTAGACAAAG-3′. The shRNA target sequence for STAT3 was 5′-GCACAATCTACGAAGAATCAA-3′.

LV-CDC37L1 and LV-NC (negative control) for overexpression of CDC37L1; LV-shCDC37L1 and LV-shNC (negative control) for knockdown of CDC37L1; Two kinds of lentiviral particles were packaged and purchased from GenePharma (Shanghai, China). Stable cell lines were established by puromycin selection after lentivirus transduction.

For generating cells stably expressing ARV7 or cells with ARV7 depletion, lentiviral particles were synthesized by GenePharm, and infected cells were selected using puromycin as the protocol recommended. The sequence for ARV7 expression is in accordance with NCBI database (NM_001348061.1) while the targeting sequence for ARV7 knockdown is 5’-GCTCCATAGCTTCCATATTGA-3’ within the 3’-UTR region. For UBE2C depletion, human UBE2C shRNA lentiviral particles (sc-61742-v) were purchased from Santa Cruz Biotechnology, and UBE2C depleted cells were selected using puromycin as well.