Penicillin and streptomycin

SH-SY5Y (ATCC Cat# CRL-2266, RRID:CVCL_0019) and HEK293T (ATCC Cat# CRL-3216, RRID:CVCL_0063) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml penicillin and streptomycin (Hyclone) at 37°C in 10% CO₂. HEK293T cells deficient for COG subunits were generated as described in (Blackburn and Lupashin, 2016 (link)). Two lines of Menkes deficient fibroblasts were used: one in conjunction with a rescue line expressing recombinant ATP7A (described in La Fontaine et al., 1998 (link)) and the other in conjunction with a familial control (Coriell GM01981 and GM01983). The former was cultured in DMEM supplemented with 10% FBS and 100 μg/ml penicillin and streptomycin at 37°C in 5% CO₂, while the latter were cultured in minimum essential media (MEM) (Thermo Fisher 11095080) supplemented with 15% FBS and 100 μg/ml penicillin and streptomycin at 37°C in 5% CO₂. Antibodies and primers can be found in Supplementary file 1. Cell were tested for mycoplasma infection using the MycoAlert Mycoplasma Detection Kit from Lonza. None of the cell lines used in this study belong to the Database of Cross-Contaminated or Misidentified Cell Lines defined by the International Cell Line Authentication Committee.

For the generation of primary bone marrow‐derived macrophages (BMDMs) from mice, tibias and femurs were flushed with IMDM media (Thermo Fisher Scientific, 12440053) supplemented with 10% of heat inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, 16000044), 50% L cell‐conditioned media, 0.6× MEM Non‐Essential Amino Acids (Thermo Fisher Scientific, 11140050), and 0.1% penicillin and streptomycin (Thermo Fisher Scientific, 15140122). Cells were cultivated at least 6 days at 37°C in a humidified atmosphere containing 5% CO₂ as previously described 8, 9, 13, 76. THP‐1 cells either expressing Cas9 or co‐expressing CASP4^−/−, CASP5^−/−, CASP4/5^−/−, and CASP1^−/− were maintained in RPMI 1640 media (Thermo Fisher Scientific, 22400105) supplemented with 10 % FBS and 0.1% penicillin and streptomycin. To induce the CRISPR system, cells were incubated with 1 μg/ml of doxycycline hyclate (Sigma, D9891) for 72 h and the knockout status was verified by immunoblotting 27. THP‐1 monocytes were differentiated into macrophages by 48‐h incubation with 25 nM phorbol 12‐myristate 13‐acetate (PMA, Sigma‐Aldrich, P8139) followed by 24‐h incubation in RPMI medium plus 10% FBS.

Jurkat human T cells (TIB-152, ATCC), HIV-DNA⁺ J-Lat full-length human T cells (clone 6.3, ARP-9846)^{64 (link)} and Raji human B cells (CCL-86, ATCC) were cultured in Gibco RPMI Medium 1640 (Thermo Fisher Scientific, 11875093) with penicillin and streptomycin (Thermo Fisher Scientific, 15140122) and 10% fetal bovine serum (FBS). Mouse fibroblasts (NIH/3T3, CRL-1658, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with penicillin and streptomycin (Thermo Fisher Scientific, 15140122) and 10% FBS. Before use, 3T3 cells were dissociated using 0.25% trypsin-EDTA (Thermo Fisher Scientific, 25200-072) and neutralized in DMEM with 10% FBS. Cell lines were used without authentication or mycoplasma contamination testing.