Flp in t rex 293 cell line

Human OATP1B1 (Uniprot: Q9Y6L6) and OATP1B3 (Uniprot: Q9NPD5) genes were codon-optimized for expression in human cells and purchased from GenScript. The synthetic genes were independently cloned in a pcDNA5 vector with a cleavable C-terminal YFP-1D4 tag. For protein expression, tetracycline-inducible stable cell lines (Flp-In™ T-REx™ 293 Cell Line, Thermo Fisher Scientific) were generated for each construct. Cells were grown and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 μg/mL streptomycin, 100 units/mL penicillin (Thermo Fisher Scientific) at 37 °C with 5% CO₂ under humidified conditions.
Before protein expression, the media was aspirated, the cells were washed once with phosphate-buffered saline (PBS) buffer, and expression medium consisting of fresh phenol red dye-free DMEM (Gibco) supplemented with 2% FBS, 100 μg/mL streptomycin, 100 units/mL penicillin, 1x GlutMAX (Thermo Fisher Scientific), and 1x Sodium Pyruvate (Thermo Fisher Scientific), was added. Protein expression was induced with 1 μg/mL tetracycline (Sigma) at 37 °C. Following 72 h of expression, cells were harvested, washed with PBS, flash-frozen in liquid nitrogen, and stored at −80 °C for a maximum of 6 months.

HEK293 cells of the FlpIn™ T-REx™ 293 cell line (ThermoFisher Scientific, USA) were targeted with CRISPR/Cas9 to induced knock-out of Gab1. Therefore, a Gab1 CRISPR/Cas9 KO plasmid (Santa Cruz, USA) coding for a pool of three different guide RNAs targeting the Gab1 gene and coding for Cas9 was transfected into FlpIn™ T-REx™ 293 cells. A Gab1 homology-directed repair (HDR) plasmid coding for a puromycin resistance flanked by a loxP site was co-transfected. Guide RNA- and Cas9-induced DNA double strand breaks activate the HDR pathway leading to directed integration of the loxP flanked puromycin resistance into the genome and thereby destruction of the genomic Gab1 coding sequence. After picking single clones and subsequent selection of puromycin-resistent cell clones, Gab1 knock-out was confirmed by RT-qPCR and Western Blot. The engineered cell line entitled HEK293 Gab1-KO is grown in DMEM, supplemented with FCS (10%), streptomycin (100 mg/l), penicillin (60 mg/l), zeocin (100 μg/ml), puromycin (5 μg/ml) and blasticidin (5 μg/ml) in a water-saturated atmosphere in the presence of 5% CO₂ at 37 °C.

HEK-293 cells (ATCC®, CRL 1573^TM) were cultured in high glucose DMEM media (Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario) at 20% O₂ at 37 °C to a confluency of 60–80%. Cells were used for transfection to silence (siRNA) and overexpress BOK, and to overexpress BOK ΔBH3 (described below). HEK-293 cells were stably transfected with GFP-hBOK using a Flp-In-T-Rex-293 cell line (ThermoFisher Scientific®) as previously described^{26 (link)}. GFP-hBOK cells lines included WT and those with the following deletions: ΔBH3, ΔTMD. BOK-ΔBH3 plasmid was obtained by deletion of residues 65–82 corresponding to the BH3 domain of WT BOK. BOK-ΔTMD lacked the complete TMD domain. BOK WT and mutant expression was induced in the transfected cell lines by Dox at 1.5 or 2.5 ng/mL for 36 h.

Generation of HEK293^HA-TDP-43 stable cell lines, expressing HA-tagged WT, E17R, S48A, S48E, M337V, and ΔCR constructs and stable cell lines was previously described [40 (link)]. Mutants A326P and M337P were generated by site-directed mutagenesis as previously described [10 (link)] using the primers listed in S1 Table. Briefly, the HEK293-derived cells Flp-In T-Rex 293 Cell Line (Thermo Fisher Scientific) were stably transfected to express HA-TDP-43 upon induction with tetracycline (1 μg/ml). Cells were grown and maintained in DMEM (Dulbecco’s Modified Eagle’s Medium–High Glucose, Corning) supplemented with 10% FBS (fetal bovine serum) and incubated in a humid atmosphere at 37°C and 5% CO₂. Expression of HA-tagged TDP-43 construct was induced at 30% confluence.

Flp-In T-REx 293 cell line (Thermo Fisher Scientific, Waltham, USA, Cat # R78007), SAL004 (Flp-In T-REx 293-derived PDHA1-mCherry-expressing; see section ‘Creation of a PDHA1-mCherry-expressing cell line’).