Polybrene

The sequence of HOXA10 CDS was obtained from the NCBI database and cloned into a lentiviral vector (pLVX-AcGFP1-N1 plasmid). The lentiviral vector and lentiviral package plasmids (psPAX2 and pMD2.G) were co-transfected into HEK-293T cells, and the medium was replaced with a fresh medium after 12 h. After culturing for 48 h, the supernatant was filtered through a 0.45 μm syringe filter and mixed with 40% PEG 8000 at 4 °C overnight. Viruses were harvested by centrifuging at 3000 xG for 40 min, and stored at -80 °C. With the help of polybrene (Genomeditech, Shanghai, China), UCMSCs at 50% confluence were transfected with the virus. Seventy-two hours post-transduction, the viral genome was integrated into the host cell genome, and the transfection efficiency was approximately 74%.

KGN cells were cultured to achieve a 30%-40% confluence, and transfected with PLVX-CMV-3×Flag-FBXO31-PGK-Puro WT (FBXO31 OE), PLVX-CMV-3 × Flag-FBXO31(g.153-327del)-PGK-Puro MT (FBXO31 △F OE), or PLVX-CMV-eGFP-PGK-Puro (Vector) lentivirus at a multiplicity of infection (MOI) was approximately five in the presence of 2 μg/ml of polybrene (Genomeditech). Two days following transfection, cells were enriched with puromycin (2 mg/ml) for seven days. Stable cell lines were examined for FBXO31 expression by qRT-PCR, WB, and green fluorescent protein (GFP) expression.

Lentiviruses for TFEB overexpression (LV-TFEB, NM_001167827.3) and ATP6V0C silencing (LV-shATP6V0C, NM_001694) Along with their corresponding negative controls were designed and constructed by GENERAL BioL (Anhui, China). HK-2 cells were transfected with specific lentiviruses (including the lentivirus carrying the mRFP-GFP-LC3 plasmid) at a multiplicity of infection (MOI) of 20 in the presence of polybrene (Genomeditech, China) for 24 h. The cells were then cultured in complete medium for two days and treated with 2.5 μg/ml of puromycin (Sigma-Aldrich, USA) to establish stable cell lines. For siRNA transfection, Lipofectamine 3000 (Invitrogen, USA) was used to transfect siTFEB or siATP6V0C (GENERAL, China) at a concentration of 100 nmol/L, or a scrambled control siRNA, into HK-2 cells in OptiMEM (Invitrogen, USA).

The PPM1B-specific (GenBank accession no. NM_177969) targeting shRNA sequences (5′-GCAAGCGTAATGTTATTGA-3′/shPPM1B-1, 5′-CACGGGTTGAAGAGATTAT-3′/shPPM1B-2, and 5′-CTGAATCCACATAGAGAAA-3′/shPPM1B-3) and a negative control shRNA sequence (5′-TTCTCCGAACGTGTCACGT-3′/shPPM1B-NC) were synthesized by GeneChem (Shanghai, China). For lentiviral infection, Cells were plated into 6-well plates and infected with either shPPM1B (MOI = 10) or shPPM1B-NC lentiviruses in the presence of 5 mg/mL polybrene (Genomeditech, Shanghai, China). After 72 h, the GFP level from the lentivirus in HepG2 cells was assessed using the fluorescence microscope. Then, the cells were harvested and measured for the posttransfection using qPCR and Western blotting. The present results suggested that the PPM1B expression from the viral vector was observed in >90% of HepG2 cells. Further, the qPCR and Western blot assay elucidated that shPPM1B-1/PPM1B-2/PPM1B-3 were effective in HepG2 cells, with approximately 40-60% knockdown efficiency. These data suggested that the lentiviral-mediated shRNA avenue could efficiently and stably weaken the PPM1B level in HepG2 cells, and knockdown efficiency of shPPM1B-1 is especially remarkable that it was chosen for further study (Figure 3S).

The lentiviral vector PGMLV‐CMV‐MCS‐EF1‐ZsGreen1‐T2A‐Puro (Genomeditech) containing ZNF683 cDNA were used for gain‐of‐function experiments. The lentiviral construct PGMLV‐HU6‐MCS‐CMV‐ZsGreen1‐PGK‐Puro encoding short‐hairpin RNA (shRNA) targeting SH2D1B and ZNF683, or the corresponding scrambled sequences as negative controls (Table S1) were obtained. Constructs were co‐transfected with packaging plasmids (TIANGEN) into HEK293 cells to obtain recombinant lentivirus, which was added to cell cultures for 24 h in the presence of 8 µg/ml polybrene (Genomeditech). To enhance the efficacies of NK cell transfection, we employed a novel spinfection protocol.³³ Briefly, NK cells were activated for 5–7 days as described above, then mixed with lentivirus at a multiplicity of infection (MOI) of 10 and 5 µg/ml polybrene, they were centrifuged at 1000 × g for 1 h at ordinary temperature subsequently. After 5 days, transfection efficacies were assessed by RT‐qPCR before each experiment.