Oligonucleotide primers

To study the mRNA expression of DNA repair genes (Supplementary Table S1) in LNCaP cells stably overexpressing AR-FL or AR-V7, cells were seeded 24 h prior to irradiation in androgen-deprived medium. Six hours after irradiation (6Gy), either protein lysates or total RNA were recovered from the cells, followed by qRT-PCR as previously described [29 (link)]. Oligonucleotide primers specific for DNA repair genes and PPIA (peptidyl-prolyl isomerase A, used as housekeeping gene) were purchased from biomers.net (Ulm, Germany). Primer sequences are provided in Supplementary Table S2. Sequence verification of the amplification products was performed with Sanger sequencing. Gene expression was measured in triplicates per gene. Relative gene expression was assessed using the ΔΔC_τ method with PPIA as a housekeeping gene (Supplementary Tables S3 and S4).

All enzymes and kits for the isolation of DNA or the purification of PCR products or restriction fragments were purchased from Thermo Scientific^TM (Life Technologies GmbH, Darmstadt, Germany) or Analytik Jena (Jena, Germany). Oligonucleotide primers were ordered from biomers.net GmbH (Ulm, Germany). Sequences were analyzed using pDRAW32 (ACACLONE software) and DNAMAN (Lynnon Biosoft) software.
Plasmids were transferred into E. coli strains by transformation using chemically competent cells (Inoue et al., 1990 (link)) and into A. aromaticum by conjugation (Wöhlbrand and Rabus, 2009 (link)). Bacterial mating and conjugational plasmid transfer were performed as described before (Wöhlbrand and Rabus, 2009 (link)), with the exception that the DAP-auxotrophic E. coli strain WM3064 was used as donor strain.

Transient transfection was performed in 293T cells using polyethylenimine-DNA complexes (Sigma Aldrich) as described previously [17 (link)]. HeLa cells were transfected by the use of Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following plasmids were used for transfection: pDsRed1-N1 (RFP, Clontech, Kusatsu, Japan), pcDNA-UL50-HA, pcDNA-UL53-Flag [18 (link)], pEXPR-IBA5-UL34 (Strep-UL34) and pCR-N-Myc-UL31 [19 (link)], pcDNA3.1-HA-BFRF1 and pcDNA3.1-Flag-BFLF2 [20 (link)]. Expression plasmids coding for C-terminal HA-tagged or Flag-tagged MCMV, EBV and VZV NEC homologs were generated by standard polymerase chain reaction (PCR) amplification of the respective template DNA produced by infected fibroblasts. MCMV (strain Smith), EBV (strain B95-8) or VZV (strain Oka) were used to generate pcDNA-M50-HA, pcDNA-M53-Flag, pcDNA-BFRF1-HA, pcDNA-BFLF2-Flag, pcDNA-Orf24-HA and pcDNA-Orf27-Flag. Oligonucleotide primers used for PCR were purchased from Biomers (Table S1, Ulm, Germany). After cleavage with the corresponding restriction enzymes, PCR products were inserted into the eukaryotic expression vector pcDNA3.1(+) (Life Technologies, Carlsbad, CA, USA).

Expression plasmids coding for several tags (HA, AU1, Flag and His) of HCMV pUL50 or pUL53 were generated by PCR amplification of the UL50 or UL53 open reading frame (ORF), using the template pCM1029 [29 (link)]. In addition to individual full-length UL50 or UL53, a fusion version (UL50::UL53), encoding aa 1-397 of UL50 and aa 1-376 of UL53, was also generated. Primers containing tag sequences were also amplified via PCR, resulted in the fusion of the ORFs to C-terminal with different tags. Vent DNA polymerase (New England Biolabs, Ipswich, MA, USA) was obtained for performing PCR with 36 cycles (denaturation for 40 s at 94 °C, annealing for 40 s at 58 °C and polymerization for 90 s at 72 °C). PCR products were cleaved with the restriction enzymes EcoRI/XhoI and were inserted into the vector pcDNA3.1 (Invitrogen). The plasmids coding of HCMV pUL50 or pUL53 expressing red (RFP) or green fluorescent protein (GFP) were inserted into vectors pDsRed1-N1 or pEGFP-N1 (both BD Clontech), respectively after cleavage with the restriction enzyme EcoRI/BamHI. Oligonucleotide primers used for PCR were purchased from Biomers (Supplementary MaterialsTable S1).