Hap1 cell line

HAP1 cell lines were obtained from Horizon Discovery. UOK257 and UOK257-2 cell lines were provided by LSS. All other cell lines were obtained from the ATCC. Catalog numbers are listed in Supplementary Table 3. Cells were cultured at 37 °C in a CellQ incubator at 5% CO₂. HEK293, Du145, J82, UMUC-3, MDA-MB-231, A549, UOK257 and UOK257-2 cells were grown in DMEM (Sigma-Aldrich), 786-O, PC3, SW780, LNCaP and H1299 cells in RPMI-1640 (Sigma-Aldrich), A498 cells in minimum essential medium (MEM; Sigma-Aldrich), Caki-1, Caki-2, HT29, T24 and RT4 cells in McCoy’s 5A medium (Sigma-Aldrich) and HAP1 cells in Isocove’s modified Dulbecco’s medium (IMDM; Gibco). Cell lines were tested for mycoplasma contamination at the early stages of the experiments.

HEK293, Neuro2a and C2C12 cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. HepG2 cells were purchased from ATCC and cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Wildtype, AK knockout and Nrk1 knockout HAP1 cell lines were purchased from Horizon Discovery (UK) and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified incubator supplied with 5% CO₂/95% air at 37°C. HAP1 AK KO cells and Nrk1 KO cells were generated by a single basepair deletion in the human AK sequence or Nrk1 sequence using CRISPR Cas technology and the deletion was verified by sequencing by Horizon Discovery. Additional cell line information can be found in Reporting Summary.

Human HAP1 cell line was purchased from Horizon Discovery (C859). The wild-type and mutated HAP1 cell lines (HAP1-CTCFdegron, HAP1-CTCFdegron-TIR1, HAP1-RPB1-AID, DDX55 and TAF5L knock-out clones) were cultured at 37°C with 5% CO2 in IMDM GlutaMAX^™ Supplement (Gibco, 31980097) with 10% FBS (Gibco, 16000069), 1% Penicillin-Streptomycin (Gibco, 15140122).
HCT116-RAD21-AID cells were a gift from Masato Kanemaki^{30 (link)}. They were cultured at 37°C with 5% CO2 in McCoy’s 5A medium GlutaMAX^™ Supplement (Gibco, 36600021) with 10% FBS (Gibco, 16000069), 1% Penicillin-Streptomycin (Gibco, 15140122).
HEK293T cell line was obtained from ATCC (CRL-3216) and maintained in DMEM (Gibco, 11995065) with 10% FBS (Gibco, 16000069), 1% penicillin–streptomycin (Gibco, 15140122).
Cell lines were routinely tested for mycoplasma infection and tested negative (MycoAlertTM Mycoplasma Detection Kit, Lonza).

Cell lines HeLa (ATCC), 293T (ATCC), and 293T Flp-In T-REx (ThermoFisher Scientific, R78007) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin at 37 °C and 5% CO₂ unless otherwise stated. The ∆fam91a1 mutant and its WT parent HAP1 cell line were purchased from Horizon Discovery, with FAM91A1-001 having been targeted using the guide GTTGATAAGATCATCGATTC. Cells were tested regularly to ensure that they were mycoplasma free (MycoAlert, Lonza). For immunofluorescence, cells were transfected with plasmid DNA using FuGENE 6 according to the manufacturer’s instructions (Promega). Cells were fixed with 4% formaldehyde in PBS and permeabilised in 0.5% (v/v) Triton-X-100 in PBS. Cells were blocked for one hour in PBS containing 20% FCS and 0.25% Tween-20 and probed in the same buffer with primary antibodies (Supplementary Table 2) and then Alexa-labeled secondary antibodies (Molecular Probes). The cells were mounted in Vectashield (Vector Laboratories) and imaged using LSM 780 (Zeiss) or TCS SP8 (Leica) confocal microscopes. For quantification of TGN46, cells and the Golgi were defined with DAPI and ZFPL1 respectively (NIS-Elements, Nikon). The Golgi mask had a minimum threshold of 18% of the mean ZFPL1 Golgi labeling to exclude out of focus Golgi.