Hela tet on cell line

HeLa Tet-On cell line (Clontech) was used in all experiments. Cells were grown in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM, Sigma-Aldrich), supplemented with 10 % Fetal Bovine Serum (FBS, Sigma Aldrich) and penicillin (100 units/ml) and streptomycin (100 μg/ml) mixture (Sigma-Aldrich) at 37 °C with 5 % CO₂. siRNAs transfections were done using Lipofectamine RNAiMAX (Invitrogen) as described [43 (link)] after cells were cultivated for 16–18 hours with starting cell count of 0.5×10⁵ cells/mL. The plasmid DNA transfections were done next day after siRNA transfections (where applicable) or after 20–24 h of cell growth with starting cell count of 2×10⁵ cells/mL using Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. When indicated, plasmid-transfected cells were treated with 10 μM of MG-132 or DMSO (control) for 8 h at 37 °C with 5 % CO_2. In experiments with inhibition of transcription, cells were treated with 8 μM of actinomycin D (Sigma-Aldrich) for different periods of time as indicated. Artificial OmpA mRNA [52 ] was added to the samples of the actinomycin D experiment before total RNA purification to use it for normalization in RT-qPCR.