Sigenome non targeting pool 2

Transfection of CFBE41o- cells with siRNA targeting human Rab5 gene (siRab5, siGENOME Human Rab5 siRNA; Dharmacon, Cambridge, United Kingdom), human Rab11A gene (siRab11 Accell Human Rab11A siRNA; Dharmacon) or non-targeting siRNA (siCTRL, siGENOME Non-Targeting Pool #2; Dharmacon) was performed using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. CFBE41o- cells were plated on collagen-coated cell culture plates and incubated with the optimized transfection mixture containing 50 nM of siRNA, at 37°C for 24 h. Next day, cells were transferred to collagen-coated Transwell filters for 5 days to allow cell polarization. Experiments were conducted 6 days after siRNA transfection.

For reverse transfection of siRNAs, pools of four different siRNAs (siGENOME Dharmacon) per target were complexed with Lipofectamine RNAiMAX (Life Technologies) in 96-well plates. HFFs and EA.hy926 cells were added at a density of 10,000 cells per well. A highly efficient HCMV-IE siRNA [62 ] (Sigma-Aldrich) served as a positive control and the siGENOME non-targeting pool #2 (Dharmacon) was used as a negative control. Duplicate wells of all conditions were prepared. Two days posttransfection, cells were infected with HCMV-TB40/E at a multiplicity of infection (MOI) 1. The next day, cells were fixed with 80% acetone and stained for viral IE antigens.

The following antibodies were used in this study at the indicated dilutions: anti-CIP2A (CST #14805; 1 : 1000), anti-GAPDH (Sigma-Aldrich G9545; 1 : 20 000), anti-GFP (gift from Laurence Pelletier, 1 : 10 000), anti-KAP1 (Bethyl A300-274A, 1 : 5000), anti-POLE3 (Bethyl A301-245A-1; 1 : 2000), anti-POLE4 (Abcam ab220695; 1 : 200), anti-Tubulin (Millipore CP06, 1 : 2000), anti-pCHK1 (S345) (Cell Signaling #2348, 1 : 1000), anti-CHK1 (Santa Cruz sc8408, 1 : 500), anti-pRPA32 (S33) (Bethyl A300-246A-3, 1 : 20 000), anti-RPA32 (Abcam ab2175, 1 : 500). The following secondary antibodies for immunoblotting were used in this study: peroxidase-conjugated AffiniPure Bovine Anti-Goat IgG (Jackson Immuno Research 805-035-180) and peroxidase-conjugated Sheep Anti Mouse IgG (GE Healthcare NA931 V). All peroxidase-conjugated secondary antibodies were used at a dilution of 1 : 5000. Protein bands were detected using the SuperSignal West Pico enhanced chemiluminescence reagent (Thermo Fisher Scientific). The following siRNAs from Dharmacon were used in this study: control, siGENOME Non-targeting Pool #2 (D-001206-14-05); POLE3, siGENOME SMARTpool (M-008460-01-0005); POLE4, siGENOME SMARTpool (M-009850-01-0005); APEX2, siGENOME SMARTpool (M-013730-00-0005). ATR inhibitors VE-821 and AZD6738 were purchased from SelleckChem.