Padtrack vector

To overexpress SIRT1 in NRK-52E cells, the cells were transfected with pAd-Track-SIRT1 plasmid or pAd-Track vector as a control (Addgene, Cambridge, MA), with Lipofectamine 3000 as manufacturer instructed (Invitrogen, USA). After 4–6 h of incubation, the cells were washed and fresh medium was added. The transfected cells were subjected to HG treatment for later research. Western blotting was performed to confirm the transfection efficiency.

The mouse albumin promoter and B-domain-deleted (BDD) human FVIII cDNA were inserted into the pAdTrack vector (Addgene, Watertown, MA, USA) combined with pCMV-EGFP to form a binary expression cassette (Figure 1). The PmAlb-BDD-FVIII-pCMV-EGFP gene therapy plasmid was amplified in 500 mL of bacterial culture and purified using an EndoFree Plasmid Maxi Kit (Qiagen, Hilden, Germany).

For overexpression in mammalian cells, Asxl1 adenovirus was generated using the pAdEasy system^{45 (link)}. Flag-tagged mAsxl1 was subcloned into the pAdTrackCMV plasmid and recombined with the pAdEasy adenovirus backbone plasmid in E. coli BJ5183. QBI-293A cells were transfected with recombinant plasmid, and the virus was amplified. The virus titer was measured using an Adeno-X Rapid Titer Kit (Clontech). For KO in mammalian cells, recombinant adenovirus-expressing shRNA was prepared. The duplex DNA effective for both human and mouse ASXL1 (Supplementary Table S5) was digested with Notl and subcloned into the digested PBS/U6 vector (Addgene). The U6 promoter-driven shASXL1 was excised and ligated into the pAdtrack vector (Addgene). The pAdTrack-U6 shASXL1 obtained was recombined with pAdEasy-1 by transformation in E. coli BJ5183. Recombinant adenovirus was produced by transfecting the recombinant plasmid into QBI-293A cells. Infection and KO efficiency were monitored by GFP fluorescence and RT-qPCR, respectively. To evaluate the optimal multiplicity of infection (MOI) for maximal infection and transgene expression, 60-mm dishes containing 2×10⁵ A549 cells were infected with adenovirus at MOIs of 50, 100, and 200 for 24 h. Adenoviral infection efficiency was assessed based on GFP expression. Asxl1 expression in A549 cells was evaluated by RT-PCR and WB.