Ptagrfp n

HEK293 (ATCC no. CRL 1573) and HeLa cells (ATCC no. CCL-2) were cultivated at 37°C and 7.5% CO₂ in Iscove's modified Dulbecco's medium (IMDM) (Thermo Fisher Scientific) containing 10% fetal bovine serum (Biochrom) and penicillin/streptomycin (Invitrogen). RFP (pTagRFP-N, Evrogen) and β-tub-GFP-constructs (pEGFP-Tub, BD Biosciences) were transfected into HEK293 cells via the DOTAP lipofection reagent (Roche Applied Science). After selection with Geneticin (G418, 0.5 mg/mL, Thermo Fisher Scientific) for 4 to 6 weeks, stable transfectants of HEK293 were subcloned, sorted, and screened via flow cytometry for RFP and β-tub-GFP expression. For the chlamydial infection experiments, the HEK293 cells and all of the generated transfectants were cultured in the presence of 5 μg/mL heparin without any antibiotics, including G418. Immortalized epithelial cells from newborn mice (MN-R cells) were obtained from the Collection of Cell Lines in Veterinary Medicine (CCLV) of the Friedrich-Loeffler-Institut (CCLV-RIE number 282). The epithelial African green monkey kidney cell line BGM was obtained from the National Reference Laboratory for Chlamydiosis of the Friedrich-Loeffler-Institut (CCLV-RIE number 136).

CREB transcription factor activation was measured using the pCREB-Luc CREB luciferase reporter vector (Signosis, Santa Clara, CA, USA). The cells were seeded at the density of 60,000 per well of 96-well plate the day before transfection and were cotransfected with the vector and the red fluorescent protein expression vector pTagRFP-N (Evrogen, Moscow, Russia) at a 1:1 ratio using the FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The day after the transfection the cells were treated with the test substances for 3 h in a fresh medium, after which the RFP fluorescence was measured, the cells were lysed and used for the luciferase activity detection.