224 Chapter 7 METHODS Cell lines Generation and culturing The Reh cell line, which lacks expression of a functional glucocorticoid and mineralocorticoid receptor, was used to generate two inducible cell line models with either a GR (NR3C1) or MR (NR3C2) construct. Gateway multisite recombination (Invitrogen) was used for gateway cloning of lentiviral expression vectors as previously described.7 The entry vectors used for RehNR3C1 or RehNR3C2 cells were (1) attL1/attR5-flanked doxycyclineinducible promotor (third generation; Clontech), (2) attL5/attL4-NR3C1 or attL5/attL4NR3C2 complementary DNA sequence, (3) attR4/attR3-flanked DDK-tag followed by a stop codon, Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE) sequence, and a constitutive spleen focus forming virus promotor, and (4) tetracycline (doxycycline)-induced transcriptional activator protein-Thosea asigna virus 2A-truncated Nerve Growth Factor Receptor reporter. Single cells were plated to acquire cell lines with significant inducibility of the constructs. After exposure to doxycycline (0,5µg/ml) for 16 hours, the inducibility of the NR3C1- or NR3C2-constructs was assessed through flow cytometry following intracellular DDK staining. For both cell lines, two clones were selected (named RehNR3C1-A, RehNR3C1-B, RehNR3C2-A and RehNR3C2-B). Both clone A cell lines had the best inducibility and were therefore primarily used in our main experiments. Cells were cultured in RPMI 1640 medium (Gibco) supplemented with 1x Glutamax, 2% fetal calf serum (FCS) and 1% penicillin-streptomycin-fungizone solution (PFS). Cytotoxicity assays Prednisolone, dexamethasone and hydrocortisone were plated in concentrations ranging from 500µM to 8.192*10-8 µM. Both RehNR3C1 and RehNR3C2 cell lines were incubated with doxycycline 0,5µg/ml for approximately 16 hours (overnight). Doxycycline-induced and noninduced cells were plated in a concentration of 0.80*106 cells/mL and subsequently exposed to steroid treatment. After four days, viability was measured by methylthiazolyldiphenyltetrazolium bromide (MTT, Sigma Aldrich). Western blotting RehNR3C1 and RehNR3C2 cells were incubated with doxycycline 0,5µg/ml for approximately 16 hours. RehNR3C1 cells were treated with 0.8µM prednisolone, 0.16µM dexamethasone or 0.032µM hydrocortisone and RehNR3C2 cells with 0.032µM prednisolone, 0.16µM dexamethasone or 0.0028µM hydrocortisone. Protein extraction and subsequent blotting procedure on Reh cells was performed as described previously.12 Primary antibodies used for western blotting were NR3C1 (3660S, Cell signaling), DDK (DYKDDDK Tag, Rabbit mAb, 2368S, Cell Signaling), BIM (#ab32158, Abcam) and β-actin (#ab6276, Abcam).
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