Puromycin ant pr

Non-proliferative lentivirus was packaged using the lentivirus packaging system purchased from the System Biosciences. Briefly, the pCDH 5aCON-Nluc G2P or pCDH 9xGAL4UAS-Nluc R2P plasmids were mixed with a pPACKH1 plasmid mix consisting of pPACKH1-GAG, pPACKh1-REV, and pVSV-G (System Biosciences), and then transfected into COS1 cells using X-tremeGENE 9 DNA Transfection Reagent (Roche, Basel, Switzerland). After 36 h, the culture medium was filtered through a 0.45 µm syringe filter and directly added to the PC3 cells not expressing PAX6. The infected cells were then cultured with 10 µg/mL puromycin (ant-pr; InvivoGen, San Diego, CA, USA) for more than 2 weeks to establish the PC3-5aNluc and PC3-9Gal4Nluc cells.

All shRNA experiments were carried out by stable lentiviral transduction. Lentiviruses were generated by transfection of HEK293T cells with pInducer10 (Trono Laboratory, N/A, Lausanne, Switzerland) or pGIPZ (Dharmacon, N/A, Schwerte, Germany) together with the packaging plasmids psPAX.2 (Trono Laboratory, Addgene 12 260) and pMD.2G (Trono Laboratory, Addgene 12259). shRNA sequences are listed in Table S1. shRNAs against Miz1 were selected as described by Fellmann. Infections were carried out using 8 μg·mL⁻¹ polybrene (107 689, Sigma). Forty‐eight hours after infection, cells were selected with 2 μg·mL⁻¹ puromycin (ant‐pr, Invivogen, Toulouse, France), and pools of selected cells were used for downstream analyses.

Dulbecco’s modified Eagle’s medium (SH30081.01) was purchased from Cytiva- Fischer scientific (Waltham, MA, USA). Earle’s Balanced Salt Solution (24010043), phosphate-buffered saline (10010015), l-glutamine (25030024), penicillin-streptomycin (15140122), sodium pyruvate (11360070), and Geneticin (10131035) were purchased from Gibco Life Technologies (Dun Laoghaire, Dublin, Ireland). Fetal bovine serum (FB-1003/500) was purchased from Biosera (Nuaille France). Hygromycin B Gold (ant-hg), Zeocin (ant-zn), and puromycin (ant-pr) were purchased from InvivoGen (San Diego, CA, USA). Bovine serum albumin (04-100-812-C) was purchased from Euromedex (Souffelweyersheim France). jetPRIME transfection buffer (712-60) and jetPRIME transfection reagent (114-07) were purchased from Polyplus (Illkirch-Graffenstaden France). Lysotracker (PHE0023) was purchased from Invitrogen (Waltham, MA, USA). Fibronectin (PHE0023) was purchased from Life Technologies (Carlsbad, CA, USA).