Tissue culture media and supplements

Tissue culture media and supplements were obtained from Invitrogen (Paisley, UK), and tissue culture plastic was purchased from TPP (Trasadingen, Switzerland). FCS (FBS) was purchased from Biosera (Boussens, France) and Sigma (Sigma, St. Louis, USA). Human serum from human male AB plasma was of USA origin and sterile‐filtered (Sigma). All chemicals, etoposide and campthotecin were purchased from Sigma (St. Louis, MO, USA) unless indicated otherwise. Antibodies used were anti‐cdc2 (Cell Signaling Technology, Beverly, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, Abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK) and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 ProSci (Poway, CA, USA); p53 (GTX100629, GeneTex, Irvine, CA, USA); p‐p53 (9286P, Cell Signaling Technology); H2AX (613301, BioLegend, San Diego, CA, USA); γH2AX (613402, BioLegend); p21(sc‐6246, Santa Cruz Biotechnology); p27 (GTX100446, GeneTex); PARP (9542T, Cell Signaling Technology); IRF3 (11904P, Cell Signaling Technology); and 53BP1 (612522, BD Biosciences, UK). pCDNA Vpx Q76A was kind gift from J. Luban (Reinhard et al, 2014).

[³H]-Diprenorphine ([³H]-DPN) (42.30 C_i/mmol) was purchased from Perkin Elmer Life Sciences (NET11212, 250μCi, Boston, MA, USA) and naloxone hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). DPDPE ([D-Pen²,D-Pen⁵]enkephalin) and dynorphin A were purchased from Neosystem. Dermorphin was prepared and purified in house as previously described [26 (link)]. Stock solutions (10 mM) of peptides were made in distilled water, while LP1 and its derivatives in DMSO and kept at −20 °C until use. The compounds were solubilized in saline at 1 μM, with the only exception of naloxone, which was at 10 μM. Successive dilutions of standard ligands were made in saline, whereas LP1 and its analogues were solubilized in HBSS (Hank’s balanced salt solution)/HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (20 mM) buffer (containing 0.005% bovine serum albumin (BSA) fraction V). Tissue culture media and supplements were from Invitrogen (Carlsbad, CA, USA) and from Cambrex Bioscience (Walkersville, MD, USA).

The reference molecules, dermorphin, Leu-enkephalin, Dynorphin-A and N/OFQ were synthesised in house (Department of Chemical and Pharmaceutical Sciences, University of Ferrara). Tritiated UFP-101 ([³H]-UFP-101) was synthesized as described previously [20 (link)]. Tritiated diprenorphine ([³H]-DPN) and [³H]-cyclic Adenosine monophosphate (cAMP) were purchased from Perkin Elmer. Naloxone was purchased from Sigma-Aldrich Co. (Dorset, U.K.). Naltrindole (Delta antagonist) and [D-Pen², D-Pen⁵]-enkephalin (Delta agonist, DPDPE) were purchased from Tocris (Abingdon, UK). Antibodies and protein ladders were purchased from Cell Signalling (Boston, MA, USA). All tissue culture media and supplements were obtained from Invitrogen (Paisley, U.K.).