Mccoy s 5a

Transfected plasmids, CAG-Grhl3 full-length-EGFP, TA-EGFP, CP2-EGFP, and Ub-like-EGFP, were made as previously described^{9 (link)}. The open reading frame clone of HaloTag-Usp39 from Kazusa DNA Research Institute (pFN21AE5640 or FHC28713), labeled with HaloTag TMR ligand, was made according to the manufacturer’s instructions (Promega). MCF7 cells were maintained in Minimum Essential Medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Invitrogen), 0.1 mM non-essential amino acid solution, and 1 mM sodium pyruvate at 37^oC in a humidified incubator with 5% CO₂. RT4 cells were cultured in McCoy’s 5a (GE Healthcare: SH30200-01) supplemented with 10% fetal bovine serum (Gibco, Invitrogen), and 1X Glutamax (Gibco: 35050-061). MBT2 cells were cultured in Minimum Essential Media (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Invitrogen), and 1X Glutamax. Sources of cell lines are indicated in Table S1.
Transfections of a total of 2.5 μg of a plasmid preparation for each 6-well culture were performed with Lipofectamine LTX reagent (Invitrogen) according to the instructions of the manufacturer. Transfections of Grhl3 siRNA (ID33752 and ID33754; Ambion) and Usp39 Dicer-substrate siRNA (hUSP39.13.1, hUSP39.13.2, and mUSP39.13.2; IDT) were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.

HCT116, SW48, DLD1, HT29, RKO, LoVo, HCT115, and SW480 human colorectal cancer cell lines and HEK293T cells were maintained in McCoy’s 5A or DMEM supplemented with 10% FBS and 100 U penicillin/streptomycin (GE Healthcare Life Sciences) at 37 °C in a 5% CO₂ incubator. All cell lines were obtained from ATCC. No mycoplasma contamination was detected.
The RNA sequences of USP14 knock-out RKO cell line are presented in the Supplemental Table 3.

We cultured A549 cell lines in McCoy’s 5A (GE Healthcare Life Sciences, Freiburg, Germany) plus 10% fetal calf serum (FCS; Thermo Fisher Scientific, Darmstadt, Germany) at 37°C. The medium contained penicillin and streptomycin. For A549 p53-Venus reporter cells [64 (link),65 (link)], selective antibiotics (400 μg/ml G418 (Carl Roth, Karlsruhe, Germany) and 50 μg/ml hygromycin (Thermo Fisher Scientific)) were added to maintain transgene expression.
For corresponding treatment of the cells, the medium was replaced with fresh one containing dimethyl sulfoxide (DMSO; Sigma-Aldrich) as a control, IKK2 inhibitor (15 μM TPCA-1, 120 μM sc-514 or 0.93 μM BMS-345541 (MedChemExpress, Sollentuna, Sweden)), 10 ng/ml or 50 ng/ml IL-6 (PeproTech, Hamburg, Germany) or 10 ng/ml TNFα (Enzo Life Science, Lörrach, Germany) 1 h before irradiating cells (if not stated otherwise) with X-rays at a dose rate of 1 Gy/26 s (250 keV, 10 mA).