Adeasy adenoviral vector system

The HA-tagged TERT adenoviral construct (HA-TERT, human, #349917A) was purchased from ABM (Richmond, BC, CAN) and multiplied by several rounds of HEK-293 cell amplification. GFP adenovirus was constructed using AdEasy Adenoviral Vector System (Agilent, La Jolla, CA, USA) and served as infection control. Cells were cracked by three freeze and thaw cycles in Tris/HCl (pH 8). DBTRG-05MG cells were infected using 30 moi of the respective viruses. RNA for qRT-PCR (primer sequences are listed in Additional file 1: Table S2) were isolated 48 h upon infection. For viability test by ATP-assay, cells were counted and seeded 48 h after virus infection. 24 h later, cells were treated with YK-4-279 and cell viability was measured after 72 h (see cell viability assay section above).

Each Epigraph immunogen was cloned into a recombinant adenoviral expression vector as previously described [31 (link)]. Briefly, the HA genes were optimized for human gene expression then cloned into recombinant replication deficient human Adenovirus type 5 (Ad5) using the AdEasy Adenoviral Vector System (Agilent, Santa Clara, CA, USA). Successful recombinants were confirmed through restriction enzyme digest and sequencing. Recombinant Ad5-HA clones were transfected, grown, and amplified by sequentially passaging viral stocks in E1 complementing 293 cells. High viral titer stocks were obtained after a final amplification step in a Corning 10-cell stack flask. High titer viral stocks were purified with two sequential CsCl gradients, then desalted with Econo-Pac 10DG Desalting Columns (Bio-Rad). Viral stocks were stored at −80 °C in Ad-tris buffer with 10% glycerol. The virus particle quantity was measured with a NanoDrop Lite spectrophotometer at OD260. Infectious units were quantified by Adeno-X Rapid Titer Kit according to manufacturer’s instructions (Takara Bio Company, San Jose, CA USA) (Supplementary Figure S1).

One siRNA (GGCCCUGUUGGAUUAUAAU, UCP4 siRNA) targeting rat UCP4 mRNA (XM_006244611.2) and a nonsense siRNA were synthesized by GenePharma (Shanghai, China).
The UCP4 expression adenovirus (Ad5-UCP4) was constructed using the Ad-Easy Adenoviral Vector System according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA). Adenoviruses were purified by ultracentrifugation in cesium chloride gradient and then quantified.

The AdEasy Adenoviral Vector System (Agilent) was used to create adenoviral vectors containing the full-length coding sequence of murine Atoh1 under the control of a cytomegalovirus promoter. To permit the identification of infected cells, we also included sequences for an internal ribosome entry site and for RFP. Viral particles were amplified and purified by CsCl-gradient centrifugation followed by dialysis (Viral Vector Core Facility, Sanford Burnham Prebys Medical Discovery Institute). Utricles were infected in 200 μL of culture medium with 10 μL of the virus at a titer of 10¹⁰ pfu/mL. Ad-RFP virus (Vector Biolabs) at the same titer was used as a control.

AGS-CEP65 and AGS mock-transfected (AGS-M) cells were generated by transfecting pcDNA3-CEP65 and pcDNA3 alone, respectively, into AGS cells using Lipofectamine^® 2000 (Invitrogen Life Technologies). The transfected cells were subsequently selected for using 600 μg/ml G418 (Sigma-Aldrich). After three weeks, pooled colonies were recovered. Recombinant adenoviruses were generated using the AdEasy Adenoviral Vector system (Agilent Technologies, Santa Clara, CA, USA), as previously described (8 (link)). Briefly, full-length CEP65 cDNA and antisense-CEP65 cDNA were cloned into the AdEasy Shuttle vector, pAdTrack-cytomegalovirus. The resultant plasmids were linearized and co-transformed into E. coli BJ5183 cells (American Type Culture Collection) with an adenoviral backbone plasmid, pAdEasy-1. Subsequently, the recombinants were selected for with kanamycin. The linearized plasmids were transfected into HEK-293 cells using Lipofectamine^® 2000 to yield the pAd-CEP65(+) and pAd-CEP65(−) viruses. The cells were infected using a multiplicity of infection (MOI) value of 10. The expression of exogenous CEP65 was detected using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis.

Recombinant adenoviruses, expressing the wild-type or LEL mutant of CD82, were generated using the AdEasy™ adenoviral vector system (Agilent Technologies, Santa Clara, CA). Briefly, CD82 cDNA was first cloned into the pShuttle vector. E. coli BJ5183 cells were co-transformed with the linearized pShuttle vector and pAdEasy-1 adenoviral vector. Q293A adenovirus packaging cells were then transfected with the resultant recombinant Ad plasmid using Lipofectamine reagent. Primary virus stocks prepared by cell freeze-thaw and subsequent centrifugation were amplified in Q293A cells and subsequently purified by ultracentrifugation in two discontinuous CsCl₂ gradients, followed by ultrafiltration. For viral infection, titrated viral stocks were suitably diluted in complete medium to obtain the desired multiplicity of infection (MOI) and added to cell monolayers. After a 24 hour incubation, the virus-containing medium was replaced by fresh medium for further analysis.

We generated the recombinant adenovirus expression vector that carries human GLO1 (rAd-GLO1). Human GLO1 cDNA was obtained from HK2 cells using the following primers: 5′-GGGGTACCATGGCAGAACCGCAGCCC-3′ (forward), 5′-ATAGTTTAGCGGCCGCTACATTAAGGTTGCCATTTTG-3′ (reverse). Recombinant adenovirus vectors were generated using AdEasy™ Adenoviral Vector System (240009; Agilent Technologies, Santa Clara, CA, USA). Briefly, human GLO1 was subcloned into the multicloning site of AdTrack-CMV shuttle vector, which contains green fluorescent protein as reporter gene, in position between Kpn I and Not I. The resultant plasmid was co-transformed into E. coli BJ5183 strain cells with the AdEasy-1 adenoviral plasmid. Recombinant bacteria were selected by kanamycin resistance, and recombination was confirmed by PacI endonuclease restriction analysis. Finally the linearized recombinant plasmid was transiently transfected into the packaging 293 cells, using TransIT®-LT1 Transfection Reagent (MIR2304; Mirus Bio LLC, Madison, WI, USA). The culture dishes were incubated at 37 °C for 7–10 days to prepare the primary viral stock. After another two cycles of amplification of recombinant adenovirus vectors in 293 cells, the adenovirus vectors were purified by precipitation with polyethylene glycol followed by two overnights dialysis. The viral titer was determined by limiting dilution assay.