PASK IBA 3+ (IBA Streptavidin Magnetic Beads custom synthesis Lifesciences), pFRED143 (Ludwig et al., 1999); pMD2. G, psPAX
PASK IBA 3+ (IBA Lifesciences), pFRED143 (Ludwig et al., 1999); pMD2. G, psPAX2, pLVTHM, pLV-tTRKRAB-red (Wiznerowicz and Trono, 2003), VSV-G, pLKO.1 (all obtained from Addgene); pSpCas9(BB)sirtuininhibitorA-GFP (Ran et al., 2013) (PX458; Addgene); pGAD-C1 and pGBD-C1 (James et al., 1996).Cell culture, transfection and stimulationHeLa (RRID: CVCL_0030), HEK-293 (RRID: CVCL_0045) and PC3 cells (RRID: CVCL_0035) were purchased from the DSMZ. U2OS cells (RRID: CVCL_0042) had been purchased from ATCC. 293-CD40 cells (RRID: CVCL_9832) were a gift from Steve Ley and L929 (RRID: CVCL_0462) a gift of Andrea Oeckinghaus. Stocks from purchased and obtained cell lines were frozen soon after maximum of three passages and re-thawed every single 4 to six weeks. Unfavorable mycoplasma status of all cell lines was verified on a regular basis employing a PCR testkit (A3744, Applichem) based on the manufacturer’s protocol. Cells were grown in RPMI (PC3) or DMEM (all other people) medium supplemented with ten fetal calf serum (FCS) and 100 U/ml penicillin/streptomycin. 293 CD40 cells had been grown and verified by Geneticin selection (Coope et al., 2002). Pools of primary HUVEC had been bought from Thermo Fisher Scientific and grown in Medium 200 supplemented with low serum growth supplement (Thermo Fisher Scientific). Experiments employing HUVEC have been carried out soon after a maximum of six passages. Murine BMDMs immortalized using J2 viurs (iBMDM) (Gandino and Varesio, 1990) had been aSchimmack et al. eLife 2017;six:e22416. DOI: 10.7554/eLife.17 ofResearch articleCell Biologygift of Andrea Oeckinghaus and grown in DMEM medium supplemented with 10 FCS and conditioned medium (10sirtuininhibitor0 L929 cell supernatant). HEK293 cells were transfected employing normal calcium phosphate precipitation protocols. U2OS and HeLa cells have been transfected employing Lipofectamine LTX and 3000 as outlined by the manufacturer protocol (Thermo Fisher Scientific). For RNA interference, HEK293, 293 CD40 and HeLa cells have been transfected with one hundred nM siRNA and Atufect transfection reagent (1,0 mg/ml) (Silence Therapeutics) and analyzed after 72 hr. HeLa and HUVE cells had been stimulated with human IL-1b (R and D systems) in concentrations ranging from 0,5 ng/ml (qRT-PCR and EMSA) to five ng/ml (endogenous co-IPs) or with human TNFa (Biomol; five ng/ml). 293-CD40 were stimulated with 0,25 mg/ml CD40 ligand (Source Bioscience). For stimulation with recombinant RANK-L (R and D systems, 150 ng/ml), PC3 cells were serum starved overnight. iBMDM have been stimulated with murine IL-1sirtuininhibitor(PeproTech; two ng/ml).Lentiviral transductionFor inducible YOD1 expression or shRNA knock-down, HeLa cells had been double-infected with lentiviruses to create a DOX-inducible expression system according to tTR-KRAB hybrid protein (Wiznerowicz and Trono, 2003). Cells were 1st infected with pLV-tTRKRAB-red SARS-CoV-2 3CLpro/3C-like protease Protein Biological Activity vector (IRES exchanged for T2A) and afterwards with pLVTHM-based transfer vectors encoding YOD1 WT, YOD1 C160S or shYOD1 sequence, respectively, plus GFP as marker. For constitutive YOD1 expression, a single infection round with all the transfer vector encoding YOD1 WT or empty transfer vector (mock) was performed. Lentivirus production and transduction was basically performed as described previously (Hadian et al., 2011), employing pMD2.G and psPAX2 as envelope and packaging plasmids. Virus was applied to HeLa cells for about 18 hr. To induce protein or shRNA expression, cells had been treated with 0,05 mg/ml DOX (Roth) for 72 hr. Transduction efficiency was ana.
