Accell non targeting sirna pool

For gene knockdown by siRNA, Accell SMARTpool system was used as previously reported.²⁸ (link) Corneal endothelial cells at 70% confluence or ex vivo corneal endothelium were transfected on six-well plate with 1.5 µmol/L Accell SMARTpool of siRNA targeting ZEB1, SP1 or SP3 (Dharmacon, Pittsburgh, PA, USA) in Accell delivery medium according to the manufacturer's instructions. Seventy-two hours after transfection, the medium was changed to medium containing FGF2. Ten days after maintaining, another transfection with 1.5 µmol/L Accell SMARTpool of siRNA targeting ZEB1, SP1 or SP3 were performed and maintained four more days with FGF2. RNA and protein levels of ZEB1, SP1, or SP3 were analyzed by RT-PCR and immunoblotting, respectively. Accell non-targeting pool siRNA (Dharmacon) was used as a negative control, and transfection efficiency was confirmed with Accell Red Cyclophilin B Control siRNA (Dharmacon).

dHepaRG cells were transfected overnight with 0.75 µl DharmaFECT 1 siRNA Transfection Reagent (GE Healthcare Dharmacon, Lafayette, CO, USA) per well of a 12‐well plate according to the manufacturer’s instructions. 20 nM Accell human APOBEC3A (200315) siRNA SMARTpool (#E‐017432‐00‐0005, GE Healthcare Dharmacon), 20 nM Accell human ISG20 siRNA SMARTpool (#E‐015994‐00‐0005, GE Healthcare Dharmacon), or 20 nM Accell non‐targeting pool siRNA (#D‐001910‐10‐05, GE Healthcare Dharmacon) was applied. Alternatively, synthetic oligonucleotides (sequences adapted from The RNAi Consortium Collection, Mission shRNA, Sigma‐Aldrich; listed in Appendix Table S1) were annealed and ligated into a HindIII‐ and SalI‐digested pEntry backbone with H1 promoter to generate adenoviral vectors following the Gateway approach (pAd/PL‐DEST Gateway Vectors and Virapower Adenoviral Expression Systems, Invitrogen, Carlsbad, CA, USA). Resulting constructs were transfected in HEK293 cells to amplify the adenoviral vector AdV‐shISG20, and infectious units (i.u.) were determined in HEK293 cells. dHepaRG cells were transduced using an MOI of 3 i.u./cell in DMEM without supplements for 2 h.