G418 sulfate

Cells were transduced with lentiviruses for Dox-inducible ectopic expression of PARP-7 (wild-type and mutant). We generated lentiviruses by transfection of the pINDUCER20 constructs described above, together with: (i) an expression vector for the VSV-G envelope protein (pCMV-VSV-G, Addgene 8454), (ii) an expression vector for GAG-Pol-Rev (psPAX2, 12260), and (iii) a vector to aid with translation initiation (pAdVAntage, Promega) into 293 T cells using Lipofectamine 3000 reagent (Invitrogen, L3000015) according to the manufacturer’s instructions. The resulting viruses were collected in the culture medium, concentrated by using a Lenti-X concentrator (Clontech, 631231), and used to infect OVCAR4 cells. Stably transduced cells were selected with G418 sulfate (Sigma, A1720; 1 mg/ml). The cells were treated with 1 µg/mL Dox for 24 hr to induce protein expression. Inducible ectopic expression of the cognate proteins was confirmed by western blotting.

The human leukemic T cell lines Jurkat, MOLT-4 and CEM-6 were kindly provided by Dr. Abelardo López-Rivas (CABIMER, Sevilla, Spain). Jurkat cells deficient in caspase-9 and caspase-9 reconstituted Jurkat cells have been previously reported [26 (link)]. Jurkat cells stably overexpressing Bcl-2 or Bcl-x_L were generously provided by Dr. Jacint Boix (Departamento de Ciencias Médicas Básicas, Universidad de Lleida, Spain). Blood samples were obtained from healthy donors by informed consent and collected into citrates tubes. Peripheral blood lymphocytes (PBL) were then isolated by Ficoll–Histopaque density gradient centrifugation (Sigma–Aldrich) and depletion of adherent monocytes by culture on plastic dishes for 1 hr at 37°C. All cell lines were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS), 1 μM l-glutamine, penicillin and streptomycin at 37°C in a humidified 5% CO₂, 95% air incubator. Bcl-2- and Bcl-x_L-overexpressing cells were maintained in culture medium with 1 mg/ml G418 sulfate (Sigma Chemical).

HepG2 (Cat No. 88065) and Hep3B (Cat No. 88064) cell lines were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea). For transient expression, 2 × 10⁵ cells per well in a 6-well plate were transfected using TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. R0532) with the indicated amounts of plasmids and an empty vector supplemented to make the final amount of cocktails equivalent. Two NTCP-expressing cell lines, HepG2-NTCP and Hep3B-NTCP, were obtained by transfection with RC210241, followed by selection with 500 μg·mL⁻¹ G418 sulfate (Sigma-Aldrich, Cat No. A1720). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (WelGENE, Gyeongsan, Republic of Korea, Cat No. LM001-05) supplemented with 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany, Cat No. FBS-22A), 100 units·mL⁻¹ penicillin G (Sigma-Aldrich, Cat P3032-25MU), and 100 μg·mL⁻¹ streptomycin (United States Biological, Salem, MA, USA, Cat No. 21865) at 37 °C in 5% CO₂ in a humidified atmosphere. Cells were treated with ATRA (Sigma-Aldrich, Cat No. R2625), pifithrin-alpha (PFT-α) (Sigma-Aldrich, Cat No. P4359), cycloheximide (CHX) (Sigma-Aldrich, Cat No. C7698), or MG132 (Millipore, Burlington, MA, USA, Cat No. 474790) as necessary, under the indicated conditions.

The cell lines HepG2 (Cat No. 88065) and Hep3B (Cat No. 88064) were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea). Stable cell lines, HepG2-NTCP and Hep3B-NTCP, were established by transfection with RC210241, followed by selection with 500 μg·mL⁻¹ G418 sulfate (Sigma-Aldrich, Cat No. A1720). Cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (WelGENE, Gyeongsan, Republic of Korea, Cat No. LM001-05) supplemented with 10% fetal bovine serum (FBS, Capricorn Scientific, Ebsdorfergrund, Germany, Cat No. FBS-22A), 100 units·mL⁻¹ penicillin G (Sigma-Aldrich, Cat No. P3032), and 100 μg·mL⁻¹ streptomycin (United States Biological, Salem, MA, USA, Cat No. 21865) at 37 °C in 5% CO₂-humidified atmosphere. For transient expression, 2 × 10⁵ cells per well in a 6-well plate were transfected using TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. R0532) according to the manufacturer’s instructions with the designated amounts of plasmids and an empty vector supplemented to make the final amount of cocktails equivalent. The cells were treated with H₂O₂ (Sigma-Aldrich, Cat No. H1009), CHX (Sigma-Aldrich, Cat No. C7698), NAC (Sigma-Aldrich, Cat No. A7250), or MG132 (Millipore, Burlington, MA, USA, Cat No. 474790), under the indicated conditions.

Cells were transfected with lentiviruses for stable ectopic expression. We generated lentiviruses by transfection of the pInducer20 constructs described above, together with an expression vector for the VSV-G envelope protein (pCMV-VSV-G, Addgene plasmid no. 8454), an expression vector for GAG-Pol-Rev (psPAX2, Addgene plasmid no. 12260), and a vector to aid with translation initiation (pAdVAntage, Promega) into 293T cells using GeneJuice transfection reagent (Novagen, 70967) according to the manufacturer’s protocol. The resulting viruses were used to infect HeLa, MCF-7, 3T3-L1, or MDA-MB-231 cells in the presence of 7.5 μg/ml polybrene 24 and 48 hr, respectively, after initial 293T transfection. Stably transduced cells were selected with 500 µg/ml G418 sulfate (Sigma-Aldrich, A1720). For inducible expression of PAR-T, the cells were treated with 1 µg/ml doxycycline (Dox) for 24 hr.