Metafectane

The human epithelial cell line derived from lung carcinoma (A549) and human embryonic kidney 293 cells (HEK293) were obtained from ATCC and grown in Iscove’s Modified Dulbecco’s Medium (IMDM) that was supplemented with 10% fetal bovine serum, 100 U of penicillin, and 100 µg of streptomycin/mL (Sigma). Transfections were performed using Metafectane (Biontex), as suggested by the manufacturer. Human adenovirus 5 (VR-5) was obtained from ATCC.
In order to determine the effect of 17-AAG on HAdV-5 replication, A549 0.8 × 10⁶ cells/well were seeded in a six-well plate and then infected with the virus at the indicated titer. The inhibitor was added at the specified time concerning infection. Cycloheximide was used at 100 μg/mL concentration and 17-AAG was 0.25 μM, unless specified otherwise.
The virus titer was measured by 50% tissue-culture infectivity endpoint (TCID₅₀) method of Reed and Muench [76 (link)].

The cMET 3′UTR plasmid was a kind gift from Dr. Stefan Eichmüller (German Cancer Research Center), and contained the cMET 3′UTR in a psi-CHECK2 vector backbone cloned downstream of an SV40 promoter-driven Renilla luciferase gene. Cells were plated at a density of 3 × 10⁴ cells/well in 96-well plates (Nunc), and were transfected after a 24 h seeding period using Metafectane (Biontex Laboratories). 10–50 nM of labelled miR-31 probe or mimic control plasmids were co-transfected with 50 ng of the psi-CHECK-2 MET 3′UTR luciferase reporter plasmid. 24 hrs after transfection, the cells were washed with DPBS and lysed with 20 μl of 1X passive lysis buffer (Promega Corporation, USA) on a rotary shaker for 30 minutes at RT. Assays were conducted in quadruplicates and repeated at least 3 times. Reporter signals were assessed with the Dual-Luciferase Reporter Assay system (Promega, USA)). 50 μl of the Luciferase Assay Reagent II solution was added directly to each well, followed by measurements of firefly luciferase, and then addition of Stop and Glo reagent and measurement of renilla luciferase activity on a microplate reader (TECAN Trading AG, Switzerland).

For live-cell studies, we used the following plasmids: GFP-tagged HP1β [42 (link)]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany). The plasmids were introduced into E. coli DH5α, and the DNA was isolated using the Qiagen Plasmid Maxi Kit (#121693; QIAGEN, Bio-Consult, Praha, Czech Republic). The cells were transfected with 2-5 μg plasmid DNA using METAFECTANE (#T020–1.0, Biontex Laboratories GmbH, München, Germany) [35 (link)].