Adeno x expression system

We produced recombinant adenovirus using the Adeno-X Expression System (Takara, Shiga, Japan) according to the manufacturer’s protocol. Recombinant adenoviruses were prepared by disrupting HEK293 cells (JCRB9068; HSRRB, Osaka, Japan) transfected with recombinant adenoviral DNA including SIRT1 or SIRT1-HY cDNA (Addgene, Cambridge, MA). All viruses were titrated using a method that measures the 50% tissue culture infectious dose (TCID₅₀). HUC-F2 cells underwent adenoviral transduction after a 1-h infection at a multiplicity of infection (MOI) of 50.

Replication-incompetent Ad5 expressing the β green fluorescence protein gene (GFP) (U55762) powered by cytomegalovirus promoter (Ad5/GFP) were prepared with Adeno-X expression system (Takara, Shiga, Japan), which included ligation of transgene-harboring pShuttle 2 and Adeno-X vectors followed by transfection into HEK293 cells. AdF35, bearing the above transgene (AdF35/GFP or AdF35/LacZ), were produced with the Adeno-X vector of which the corresponding genomic fragment (AY271307 at 30827–33609) was replaced with that of the Ad35 DNA (Avior Therapeutic, Seattle, WA, USA). These replication-incompetent Ad5 and AdF35 vectors used the same cytomegalovirus promoter (BK000394) to activate the respective genes. Replication-competent Ad5 or AdF35 in which the E1 gene was activated by an exogenous regulatory element, Ad5/Sur, Ad5/MK, Ad5/COX-2, AdF35/Sur, AdF35/MK and AdF35/COX-2, were prepared by replacing the authentic E1 promoter region with 5′ upstream regulatory sequences of the MK (0.6 kb, D10604) [12 (link)] the Sur (0.5 kb, U75285) [13 (link)], or COX-2 (0.3 kb, U04636) gene [14 (link)]. Ad were purified with an Adeno-X virus purification kit (BD Biosciences, San Jose, CA, USA) and the numbers of virus particles (vp) per ml was estimated with the formula, absorbance at 260 nm of purified Ad in the presence of 0.1% sodium dodecyl sulfate × 1.1 × 10¹².

Replication-competent Ad-delE1B, Ad-p53 or Ad expressing β-galactosidase gene (Ad-LacZ) were prepared with an Adeno-X expression system (Takara, Shiga, Japan). Virus particles (vp) were estimated with the formula: absorbance at 260 nm in 0.1% sodium dodecyl sulfate × 1.1 × 10¹².

sparc −/− mice were a gift from Dr. Neveen Said at University of Virginia, Charlottesville, Virginia, USA^{27 (link)} and were maintained in the accredited pathogen-free Second Xiangya hospital mice facility on a 12 h light/dark cycle^{28 (link)}. C57BL/6 mice were purchased from Model Animal Research Center of Nanjing University. All experiments were approved by the Animal Care Research Committee of Second Xiangya Hospital (ACRCSXH) and carried out as per ACRCSXH guidelines.
Human sparc cDNA clone was described by us before^{29 (link)} and subcloned into pShuttle vector (Clontech). Adenovirus expressing human SPARC was constructed using Adeno-X expression system (Clontech) as described before^{30 (link),31 (link)}. GAPDH, Horseradish peroxidase labeled donkey anti rabbit or donkey anti mouse antibodies were from Cell Signaling (Beverly, MA). Recombinant mouse SPARC protein (cat. number 942-SP-050) was purchased from R & D systems. Oxotremorine M (Oxo-M) and LY294002 were purchased from Sigma-Aldrich. CCG4986 was purchased from ChemBridge (San Diego, CA).

HTNV strains 76–118 were provided from our library. The Adeno-X™ expression system including the pshuttle vector and Adeno-X™ viral DNA was obtained from Clontech (Mountain View, CA, USA). Purified GP was obtained from the Lanzhou Biological Product Academy, and the NP was expressed and purified by our laboratory. The human embryonic kidney (HEK) cell line 293(obtained from the ATCC, Rockville, MD, USA) was used for packaging and propagating of the recombinant adenoviruses, and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA), which was supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). Vero E6 cells (Vero C1008, ATCC CRL 1586) used for the cellular microculture neutralization test, and B16 murine melanoma cells (B16F10, ATCC) were used for establishing target cells for the CTL assay, and were maintained in RPMI-1640 (Invitrogen, Carlsbad, CA, USA), which was supplemented with 10% fetal calf serum (FCS) (Gibco). All cells were incubated at 37°C in an atmosphere of 5% CO₂ in 95% air.

MiR-33a-5p mimics (mimics-miR-33a-5p), miR-33a-5p inhibitor, and their negative controls (miR-NC and inhibitor-NC, respectively) were designed and synthesized by RiboBio (Guangzhou, China). Small interfering RNAs (siRNAs) targeting USP46 and circ-NNT (si-USP46 and si-circ-NNT, respectively) and a scrambled form used as control (shRNA NC) were obtained from Dharmacon (Lafayette, CO, USA). Mimics-miR-33a-5p, mimics-NC, miR-33a-5p inhibitor, inhibitor-NC, si-USP46, circ-NNT shRNA, and shRNA NC were transfected into cultured cardiomyocytes using Lipofectamine® 2000 (Invitrogen) following manufacturer’s instructions. The circ-NNT exon along with the endogenous flanking sequence (1 kb upstream) was inserted into the pcDNA3.1 vector, and part of the upstream flanking sequence was inserted in an inverted orientation downstream. The mouse USP46 was cloned by PCR using mouse cDNA as the template and inserted into the pcDNA3.1 vector. All vectors (pcDNA-circ-NNT, pcDNA-USP46, and pcDNA3.1 empty vector) as well as si-USP46, circ-NNT shRNA, and shRNA NC were cloned into the Adeno-X Expression System (Clontech, Otsu, Japan) following manufacturer’s instructions. All adenoviral constructs were amplified in HEK293 cells. Adenoviral infection of HEK293 cells or cardiomyocytes was carried out as previously described [39 (link)].

Adenoviruses were constructed using the Adeno-X™ expression system (Clontech Laboratories). Briefly, pAdeno-X/Flag-Vpr-IRES-ZsGreen1 and the control pAdeno-X/IRES-ZsGreen1 were digested with PacI. The linear adenoviral DNA was then transfected into low-passage 293 cells. Adenovirus generation was confirmed by the appearance of a cytopathic effect and by the expression of ZsGreen1 protein. The adenoviruses were harvested and viral titers were determined on low-passage 293 cells using Adeno-X™ Rapid Titer Kit (Clontech Laboratories).
For adenovius infection, target cells were seeded in 24- or 6-well polystyrene plates (TPP Techno Plastic Products AG) or 35 mm glass-bottom dishes (IWAKI, Tokyo, Japan). On the following day, the culture medium was removed and the cells were infected at MOI 50 or 100.