Macsfectin

HeLa (human cervix carcinoma cells) and HEK 293T (human embryonic kidney) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, Karlsruhe, Germany) with high glucose, supplemented with 10% fetal calf serum (FCS, Gibco), 100 U/mL Penicillin and 100 µg/mL Streptomycin (Sigma-Aldrich, Taufkirchen, Germany) at 8% CO₂ and 37 °C.
Twenty-four hours prior to transfection cells were seeded onto 12-well plates. For transfections, 1 µg genome editing plasmid was transfected using MACSfectin™ (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. After 24 h, cells were counted and seeded into 96 well plates (1 cell/well). Wells containing single-cell clones were expanded and used for further analysis.

The coding region of SLC35A1 was PCR amplified from HEK293T cells and cloned in-frame using XbaI and BamHI digestion into pExpr-IBA103 (IBA, Göttingen, Germany) with a C-terminal Twin-Strep-tag. (For primer sequences, see Table 1.) All constructs were verified by sequencing (Seqlab, Göttingen, Germany). For transient transfections, the cells were seeded onto 12-well plates 24 h before transfection, and 500 ng of plasmid DNA was transfected using MACSfectin™ (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. The following day, the cells received the virus and were harvested 48 h post-transduction in lysis juice (PJK, Kleinblittersdorf, Germany).

Second-generation CD33-CAR incorporating the My96 scFv sequence was constructed as described earlier [22 (link)]. Briefly, the My96 scFv was combined in frame with CD8 hinge and transmembrane domain, 4-1BB/CD137 co-stimulatory domain, and CD3ζ activation domain. A leader peptide derived from GM-CSFRα was included to facilitate CAR cell surface expression. Third-generation self-inactivating baboon envelope-pseudotyped lentiviral vectors (BaEV-LVs) were produced by transient transfection into HEK293T cells using MACSfectin (Miltenyi Biotec) or polyethylenimine (PEI) [23 (link)].

AGA cDNAs from HeLa cells (wildtype AGA), from AGU-Fin and T122K-fibroblasts were amplified by PCR and cloned into pcDNA3 (Invitrogen, Thermo Fisher Scientific, Karlsruhe, Germany) using BamHI and XhoI and by XbaI and BamHI into pExpr-IBA103 (IBA, Göttingen, Germany) with a C-terminal Strep-tag. Primer sequences were:
AGA-pcDNA3-fwd: 5′-CTATAGGATCC ATGGCGCGGAAGTCGAACTTG-3′
AGA-pcDNA3-rev: 5′-CTATA CTCGAG TTA GATGCAGTCCACTTTTTCC-3′
AGA-IBA-fwd: 5′-CTATATCTAGA ATGGCGCGGAAGTCGAACTTG-3′
AGA-IBA-rev: 5′-CTATAGGATCC GATGCAGTCCACTTTTTCCTC-3′.
R116W amino acid substitution was produced by site-directed mutagenesis of the WT AGA construct. Correctness of all constructs was verified by sequencing.
24 h prior to transfection, the cells were seeded onto 12-well plates. For transfections, 400 ng of expression plasmids (either 400 ng in case of a single plasmid or 2 × 200 ng for cotransfection of two plasmids) were transfected using MACSfectin™ (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. After 24 h, cells were transferred onto 6-well plates and harvested 48 h post transfection in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40), supplemented with protease inhibitor cocktail for 30 min on ice. Protein concentrations were determined according to Bradford. Lysates were used for Western Blot.

The anti-p53 antibodies Pab421, Pab246, CM5 and 1C12 were purchased from Millipore (Darmstadt, Germany), Vector laboratories (Burlingame, CA, USA) and Cell signaling (Danvers, MA, USA), respectively. From Santa Cruz (Dallas, TX, USA), we obtained the antibodies against Oct-4 (C-10) Nanog (C-4), c-Jun (H79) and PCNA (PC10). Antibodies against acetylated and phosphorylated p53 (K379, S6, S15, S392) were from Cell signaling. Antibodies against GAPDH (6C5), PARC (PO69) and α-7 (MCP72) were from Hytest (Turku, Finland), BioLegend (San Diego, CA, USA) and Enzo (Loerrach, Germany), respectively. Antibodies against β-Actin and Histone H3 were purchased from Abcam (Cambridge, UK) and the antibody against MdmX (MDMX82) was from Sigma-Aldrich.
siRNAs were purchased from Eurofins (Ebersberg, Germany). Sequences are available on request. siRNA transfections were performed using Macsfectin (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer's instructions.

For knocking out the expression of the sialic acid transporter SLC35A1 in HEK293T wild-type, HEK293T-AGA-knockout [19 (link)], and HeLa wild-type cells, the guide RNAs (gRNAs) were designed with the E-Crisp design tool (DKFZ, Heidelberg, Germany) and cloned via BbsI into pSpCas9(BB)-2A-Puro (PX459, Addgene plasmid #48139, a kind gift of Feng Zhang) [20 (link)]. The gRNA sequences are shown in Table 1. Twenty-four hours before transfection, cells were seeded onto 6-well plates. For transfections, 2.5 µg genome editing plasmid was transfected using MACSfectin™ (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. After 24 h, the cells received a culture medium containing puromycin (2 µg/mL) for 24 h to eliminate untransfected cells. Thereafter, the cells were counted and seeded into 96-well plates (1 cell/well). Wells containing single-cell clones were expanded and used for further analysis. For analysis, cells were lysed with the Phire™ animal tissue direct PCR kit (Thermo Fisher Scientific, Dreieich, Germany). Genomic DNA was PCR amplified. The knockout was confirmed by sequencing the PCR products (Microsynth Seqlab, Göttingen, Germany). Primer sequences are shown in Table 1. Lack of protein expression was confirmed by immunofluorescence staining for SLC35A1.