U2OS, U2OS derived and HEK293 cells were cultured at 37°C in a humidified
atmosphere containing 5% CO2 in Dulbecco’s Modified Eagle
Medium (DMEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum
(FBS) from BioSera or – for U2OS cells stably expressing tetracycline
repressor (U2OS Trex) and derived inducible stable cell lines –
tetracycline negative FBS from PAA Laboratories, 100 U/ml penicillin, and 100
µg/ml streptomycin (Sigma-Aldrich). Additional supplements were used to
maintain the following stable cell lines: 0.5 mg/ml G418 (Invitrogen) to select
constitutively stably expressing GFP-CtIP4 (link)
and GFP-FLAG-MRE11 U2OS cells; 2 µg/ml blasticidin (Invitrogen) for U2OS
Trex cells, 2 µg/ml blasticidin (Invitrogen) and 0.2 mg/ml zeocin (Life
technologies) for U2OS Trex cells stably expressing inducible GFP or siRNF138-1
resistant GFP-RNF138 (WT and RM), 2 µg/ml blasticidin (Invitrogen) and
0.5 mg/mL G418 (Invitrogen) for U2OS Trex cells stably expressing inducible
siALL-Ds resistant UBE2D1 (WT and C85S). 2 µg/ml puromycin
(Sigma-Aldrich) for U2OS TLR cells. Doxycycline (Sigma-Aldrich) was added at
final concentrations of 0.001 µg/ml, 0.003 µg/ml or 1-2
µg/ml for 24-48 hours to induce comparable levels of GFP, GFP-D1-CD or
GFP-D1-WT/GFP-RNF138-WT/GFP-RNF138 C18/21A expression, respectively.
atmosphere containing 5% CO2 in Dulbecco’s Modified Eagle
Medium (DMEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum
(FBS) from BioSera or – for U2OS cells stably expressing tetracycline
repressor (U2OS Trex) and derived inducible stable cell lines –
tetracycline negative FBS from PAA Laboratories, 100 U/ml penicillin, and 100
µg/ml streptomycin (Sigma-Aldrich). Additional supplements were used to
maintain the following stable cell lines: 0.5 mg/ml G418 (Invitrogen) to select
constitutively stably expressing GFP-CtIP4 (link)
and GFP-FLAG-MRE11 U2OS cells; 2 µg/ml blasticidin (Invitrogen) for U2OS
Trex cells, 2 µg/ml blasticidin (Invitrogen) and 0.2 mg/ml zeocin (Life
technologies) for U2OS Trex cells stably expressing inducible GFP or siRNF138-1
resistant GFP-RNF138 (WT and RM), 2 µg/ml blasticidin (Invitrogen) and
0.5 mg/mL G418 (Invitrogen) for U2OS Trex cells stably expressing inducible
siALL-Ds resistant UBE2D1 (WT and C85S). 2 µg/ml puromycin
(Sigma-Aldrich) for U2OS TLR cells. Doxycycline (Sigma-Aldrich) was added at
final concentrations of 0.001 µg/ml, 0.003 µg/ml or 1-2
µg/ml for 24-48 hours to induce comparable levels of GFP, GFP-D1-CD or
GFP-D1-WT/GFP-RNF138-WT/GFP-RNF138 C18/21A expression, respectively.
