Blasticidin

HEK293T cells for pseudovirus production and HEK293T_ACE2 cells for pseudovirus neutralization experiments were cultured at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich) and 5 μg/ml Gentamicin (Sigma-Aldrich). The medium for the 293T_Ace2 cells additionally contained 5μg/ml Blasticidin (GIBCO). For constitutive expression of ACE2 in 293T cells, a cDNA encoding ACE2, carrying two inactivating mutations in the catalytic site (H374N & H378N), was inserted into CSIB (Kane et al., 2016 (link)) 3′ to the SFFV promoter (Robbiani et al., 2020 (link)). 293T_ACE2 cells were generated by transduction with CSIB based virus followed by selection with 5 μg/ml Blasticidin (GIBCO). Expi293F cells (GIBCO) for protein expression were maintained at 37°C and 8% CO₂ in Expi293 Expression medium (GIBCO), transfected using Expi293 Expression System Kit (GIBCO) and maintained under shaking at 130 rpm. The gender of the HEK293T, HEK293T_ACE2 and Expi293F cell lines is female. Cell lines were not specifically authenticated.

mES cells (CreERT2+, EGFP+) were infected with lentiviruses encoding sgEGFP1 and Cas9-p2a-blasti (kind gift from Julian Jude) or sgEGFP1 Pulse-Switch construct. Cells were selected with blasticidin (Gibco, 5 µg/ml final concentration) for 4 days starting at day 1 p.i. Subsequently, the cultures were split into 2 sets, one set was kept under blasticidin selection and in the second set, CreERT2 was induced with 4OH-tamoxifen (Sigma–Aldrich, 0.5 µM final concentration) for 48 h. On day 12 p.i. EGFP loss was measured with flow cytometry (BD Fortessa).
WT NIH 3T3 mouse embryonic fibroblasts were infected with CreERT2-GFP retrovirus and subsequently with a lentivirus encoding EBFP2. Cells were further sorted for GFP and EBFP2 and expanded clonally. Resulting NIH3T3 cells (CreERT2+, GFP+, EBFP2+) were infected with lentiviruses encoding sgEGFP1 and Cas9-p2a-blasti or with sgEGFP1 Pulse-Switch construct in 24-well format. Cells were selected with blasticidin (Gibco, 5 µg/ml final concentration) for 2 days starting at day 1 p.i. Subsequently, the cultures were split into two sets of plates, one set was kept non-induced and in the second set, CreERT2 was induced with 4OH-tamoxifen (Sigma–Aldrich, 0.5 µM final concentration) for 4 days. On day 10 p.i. (day 7 post induction) GFP and EBFP2 loss was measured with flow cytometry (BD Fortessa).

Two independent guide RNAs for ITGA5 (Table S2) from the Brunello sgRNA library (49 (link)) were synthesized (Sigma-Aldrich) and cloned in to the pLentiCRISPRv2-blast plasmid (#98293; Addgene). After confirmation by Sanger sequencing, each plasmid was used to generate a lentivirus. Lentiviral packaging was carried out in HEK293T cells using the third-generation packaging plasmids pCMV delat R8.2 (#12263; Addgene) and pCMV-VSVG (#8454; Addgene). A549 or HSAE cells were transduced with the lentiviruses in the presence of 10 µg/mL polybrene (Santa Cruz Biotechnology). The next day, transduced cells were selected with 20 µg/mL blasticidin (A11139-03; Gibco) and the blasticidin-resistant cells were processed for single colonies. Independent single-cell-derived gene-deletion cell lines were confirmed by Western blotting with an anti-integrin α5 antibody (AB150361; Abcam).

A rat PC12 cell line overexpressing human A53T mutant α-syn was constructed using a pEGFP-SNCA-A53T recombinant plasmid (kindly provided by Dr Stephanie Cobb, Mayo Clinic, FL, USA) and the lentiviral gene transfer method. Transfected PC12 cells were further screened with 5 μmol/l blasticidin (Invitrogen Life Technologies, Carlsbad, CA, USA) and obtained using a limiting dilution assay. Stably transfected PC12 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated horse serum (Gibco-BRL), 5% (v/v) fetal bovine serum (Gibco-BRL) and blasticidin (5 μmol/l). Cells were cultured at 37°C in humidified air with 5% CO₂. All experiments were performed 24–48 h after cell seeding.