Pog44 vector

To produce stable cell lines stably expressing MAC-tagged preimplantation factors, Flp-In T-REx 293 cells (Invitrogen, Life Technologies, R78007, cultured in manufacturer’s recommended conditions) were co-transfected with the expression vector and the pOG44 vector (Invitrogen) using Fugene6 transfection reagent (Roche Applied Science). One day after transfection, cells were selected in 1% Streptomycin-Penicillin and 100 µg/ml Hygromycin for two weeks after which positive clones were pooled and amplified. Green fluorescent protein (GFP) tagged with MAC-tag was used as a negative control and processed parallel to the bait proteins.
Stable cell line was expanded to 80% confluence in 20 × 150 mm cell culture plates. Ten plates were used for AP-MS, in which 2 µg/ml tetracycline was added for 24 h induction, and ten plates for BioID, in which 50 µM biotin in addition to tetracycline, was added for 24 h before harvesting. An exception to this was DUX4, which was cultivated for 20 h, respectively, due to DUX4 induction killing a large number of cells. Cells from five fully confluent dishes were pelleted as one biological sample. In total two biological replicates in two different approaches were produced. Samples were snap frozen and stored at –80 ˚C.

UBASH3B and LUBAC expression plasmids were generated by enzymatic LR clonase reaction (Invitrogen) of a UBASH3B pDONR vector (Orfeome v5.1) and a pTO-SH entry vector, encoding a C- or N-terminal Strep-HA tag. The UBASH3B expression vector and pOG44 vector (Invitrogen) were cotransfected in HEK Flp-In 293 T-Rex cells (Invitrogen) using X-tremeGENE transfection reagent (Invitrogen). Two days after transfection, cells that had undergone recombination were selected using DMEM supplemented with 100 μg/mL of hygromycin (Invitrogen) and 19 μg/mL of blasticidin (Huberlab) for 2 to 3 wk.

To produce stable cell lines stably expressing MAC-tagged LEUTX, Flip-In T-REx 293 cell lines (Invitrogen, Life Technologies, R78007, cultured in manufacturer’s recommended conditions) were co-transfected with the expression vector and the pOG44 vector (Invitrogen) using Fugene6 transfection reagent (Roche Applied Science). One day after transfection, cells were selected in 1% Streptomycin and 100 μg/ml Hygromycin for two weeks after which positive clones were pooled and amplified. Green fluorescent protein (GFP) tagged with MAC-tag was used as a negative control and processed parallel to the bait proteins. Stable cell line was expanded to 80% confluence in 20 × 150mm cell culture plates. Ten plates were used for AP-MS, in which 2 μg/ml tetracycline was added for 24 h induction, and ten plates for BioID, in which 50 μM biotin in addition to tetracycline, was added for 24 h before harvesting. Cells from five fully confluent dishes were pelleted as one biological sample. In total two biological replicates in two different approaches were produced. Samples were snap frozen and stored at –80°C.

T‐REx™ Flp‐In cells were co‐transfected with the corresponding expression plasmid and the pOG44 vector (Invitrogen) encoding the Flp‐recombinase using jetPrime (Polyplus) according to the manufacturer's instructions. Two days after the transfection, cells were selected in hygromycin (100 μg/ml) and blasticidin C (15 μg/ml) containing medium for 3 weeks.

Flp‐In™ T‐REx™ 293 cell lines (Invitrogen, Carlsbad, CA, USA) were cultured according to the manufacturer's instructions and utilized for generating stable cell lines that expressed the gene of interest with an inducible promoter. Cells were co‐transfected with the expression vector and the pOG44 vector (Invitrogen) using the FuGENE 6 transfection reagent (Roche Applied Science, Penzberg, Germany). Four days after transfection, the cells were put in 100 μg/ml hygromycin selection media for 2 weeks. Positive clones were then pooled and amplified. Stable cell lines were each expanded to 80% confluence in 20 × 145 mm cell culture plates. Ten plates were used for the AP‐MS approach and ten for the BioID experiments. Expression of the gene of interest was induced 24 h before harvesting the cells with 1 μg/ml tetracycline. For the BioID plates, 50 μM of biotin was added. Cells from five plates were pelleted as one biological sample. Therefore, each bait protein had two biological replicates in both approaches. The samples were snap‐frozen and stored at −80°C.

Flp-In 293 T-REx cells (HEK293 parental, Invitrogen) containing a single genomic FRT site and stably expressing the Tet repressor were grown in Dulbecco’s modified Eagle medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco, Thermo Fischer Scientific) and 50 μg/ml Normocin (ant-nr-2, InvivoGen).
Flp-In INS1 #5-3.19 cells (INS1 parental) were a gift from G.Ryffel and S.Senkel and have been described elsewhere (55 ). INS1 parental cells were grown in RPMI-1640 media supplemented with 10% FBS, 1 mM sodium pyruvate (S8636, Sigma-Aldrich), 10 mM HEPES pH 7.2, 2 mM L-glutamine (25030-024, Gibco, Thermo Fisher Scientific), and 50 μM beta-mercaptoethanol (31350-010, Gibco, Thermo Fisher Scientific). For targeted integration of MANF, both HEK293 and INS1 parental cells were transfected with pcDNA5/FRT/TO pre-SH-MANF or pcDNA5/FRT/TO GFP-SH expression plasmids and pOG44 vector (Invitrogen) for co-expression of the Flp-recombinase using FugeneHD (E2311, Roche) transfection reagent. Two days after transfection, the stable cell lines were selected with 50 μg/ml Hygromycin-B Gold (ant-hg-1, InvivoGen) for 2 weeks.