Calcium phosphate transfection reagent

S2 cells originating from D. melanogaster were cultured in Schneider's medium supplemented with 10% fetal calf serum. ML-DmD9 cells originating from the wing disc of D. melanogaster were purchased from DGRC and cultured in M3 medium supplemented with 5% fetal calf serum, BPYE medium and 10 µg/ml insulin. These cells were transfected with DNA using calcium phosphate transfection reagent (Thermo Fisher Scientific). Stable transformants were selected using 300 µg/ml hygromycin and cloned. Protein expression was induced by incubating cells in the presence of 0.5 mM CuSO₄ for 24 h. To knock down PIG-B-interacting proteins, double-stranded RNA for each gene and GFP (as a control) was synthesized using a T7 In Vitro Transcription Kit (TAKARA) and transfected into S2 and ML-DmD9 cells using calcium phosphate transfection reagent (Thermo Fisher Scientific). The primers used to generate double-stranded RNA are listed in Table S4. CHO-K1 cells (F21.3.8) were cultured in Ham's F-12 medium and transfected with plasmids using Lipofectamine 2000 (Thermo Fisher Scientific).

The plasmids from Addgene were used for lentiviral transduction. pMD2.G and psPAX2 were gifts from Didier Trono (Addgene plasmids #12259 and #12260, respectively). pHAGE N-mRuby3 (from SARS-CoV-2) IRES puro was a gift from Raphael Gaudin (Addgene plasmid #170466). Recombinant lentiviral particles were produced by cotransfection of HEK293T cells in a T25 flask with 2.3 μg pMD2.G, 4.3 μg psPAX2, and 10.2 μg pHAGE N-mRuby3 (from SARS-CoV-2) IRES puro using Calcium Phosphate Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The virus suspension was collected 72 h after transfection. The lentivirus was concentrated using the Lenti-X concentrator (Takara Bio, Otsu, Japan) according to the manufacturer’s protocol. Human cancer cell lines A549 and A431 were seeded in 24-well plates (1 × 10⁴ cells/well) one day before viral infection. For lentivirus transduction, lentiviral particles were suspended in a culture medium containing 10 μg/mL polybrene (Sigma-Aldrich, Burlington, MA, USA). Stable cell lines were selected with puromycin (1 μg/mL, Acros Organics, Geel, Belgium).

S2 cells originating from D. melanogaster were cultured in Schneider's medium supplemented with 10% fetal calf serum (FCS). ML-DmD9 cells originating from a wing disc of D. melanogaster were purchased from DGRC and cultured in M3 medium supplemented with 5% FCS, 0.05% KHCO₃, 0.1% yeast extract and 0.25% bactopeptone (BPYE) and 10 µg/ml insulin. DNA transfection into these cells was performed with the calcium phosphate transfection reagent (Thermo). Protein expression was induced by incubating cells in the presence of 0.1 mM CuSO₄ for 6 h. To knock down DmPIG-O, double-stranded RNAs corresponding to nucleotides 2637–2889 of DmPIG-O and the entire region of GFP (as a control) were synthesized using a T7 in vitro transcription kit (TAKARA) and transfected into ML-DmD9 cells.
The PIG-B-deficient cell line (CHO1.33) was generated by chemical mutagenesis of the parental CHO F21 cells and selection using the GPI-anchor-binding toxin aerolysin (Ashida et al., 2005 (link)). The PIG-B-deficient cell line (CHO1.33) is referred to as class B cells in the text. CHO-K1 cell lines (F21.3.8) stably expressing human CD59 and DAF, as well as class B cells were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum, 600 µg/ml G418 and 400 µg/ml hygromycin. Plasmids were transfected into cells using Lipofectamine 2000 (Thermo).