HeLa cells were maintained in DMEM containing 10% fetal bovine serum and penicillin/streptomycin at 37°C, 5% CO2. Transient transfections were performed using the X-tremeGene transfection reagent (Roche). Reporter constructs were stably integrated into the FRT site of a HeLa cell line containing a tetracycline repressor. Expression of the reporter proteins 2×RFPtevSUN2-GFP, 2×RFPtevSUN2(1–260)-GFP, 2×RFPtevLBR(fl)-GFP, and 2×RFPtevGFP-LBR(1–245)-GFP was induced for 16 h by the addition of 200 ng/ml tetracycline. Expression of 2×RFPtevLBR(1–245)-GFP and 2×RFPtevLAP2β-GFP was induced for 16 h by 20 ng/ml. AR proteins were induced for 8 h with 200 ng/ml tetracycline in HeLa cell lines constitutively expressing H2B-Plum-FKBP. The HeLa cell line (Mitchell et al., 2010 (link)) stably expressing rat POM121-3×GFP (Rabut et al., 2004 (link)) from the pEGFP backbone (CMV promoter; BD) has been described previously.
For depletion of cellular ATP (and GTP; Schwoebel et al., 2002 (link)), cells were treated with 6 mM deoxyglucose (Sigma-Aldrich) and 10 mM NaN3 in glucose-free DMEM containing 0.5% dialyzed FCS. Analysis started 15 min after addition of the energy depletion medium.
For depletion of cellular ATP (and GTP; Schwoebel et al., 2002 (link)), cells were treated with 6 mM deoxyglucose (Sigma-Aldrich) and 10 mM NaN3 in glucose-free DMEM containing 0.5% dialyzed FCS. Analysis started 15 min after addition of the energy depletion medium.
