Blasticidin s hydrochloride

Arid1a^_/− and Nsd2^+/− cells were generated by CRISPR/Cas9 system in the RAW264.7 cell line. Briefly, the Arid1a^_/_ RAW264.7 cells were transfected with Cas9 and pGL3-U6-sgRNA plasmid, then added blasticidin S hydrochloride (2 μg/mL; Life Technology) and puromycin (3 μg/mL; Life Technology) after 24 h. Nsd2^+/− RAW264.7 cells were transfected with sgRNAs–targeted-Cas9/green fluorescent protein (PGX458) plasmid. Single transfected cells were sorted. The clones were detected by PCR. The sequence of sgRNA are listed as follows: Arid1a- A1 up: 5’-CACCCTGGTTATAGTATGGAGTC-3’; Arid1a -B1 up: 5’-CACCCTGTTGTGTGGTGGACTGC-3’; Nsd2-AW1 up: 5’-CACCAAATCCTTGGCAGTGCAAA-3’; Nsd2- BW1 up: 5’- CACCACTAGGAGGAACAGGAAG-3’.

The cell lines used in this work were L. braziliensis M2903 (MHOM/BR/75/M2903) and its genetically modified derivatives. Promastigotes were cultivated at 25°C in M199 medium (Sigma-Aldrich) supplemented with 2.2 g/L NaHCO₃, 0.005% hemin, 40 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) pH 7.4, 10% heat-inactivated fetal calf serum (FCS) (Gibco™), and 0.2 µg/ml biopterin (Sigma-Aldrich). The appropriate selection drugs were added at 32 µg/ml hygromycin B (Gibco™), 5 µg/ml blasticidin S hydrochloride (Gibco™), and 20 µg/ml puromycin dihydrochloride (Gibco™).

NCI-H23 (aka H23) cells were obtained from ATCC (CRL-5800) and ACE2 lentiviruses were filtered through a 0.45 μm filter and used to transduce H23 cells using reverse transduction. Briefly, filtered virus particles are added to the H23 cell suspension with RPMI 1640 (Gibco Cat# 1185093) media supplemented with 10% FBS and 8 μg/mL polybrene (Sigma Cat# TR-1003-G). 72 h post transduction, media was changed to RPMI 1640 supplemented with 10% FBS and 4 μg/mL blasticidin S hydrochloride- (Gibco Cat# R21001). Cells were expanded in increasingly larger cell culture plates and ACE2 expression was confirmed by infecting with SARS-CoV-2 (strain Canada/ON/VIDO-01/2020) and flow cytometry. Single clone isolation from the H23-ACE2 cell pool was carried out by the array dilution method in 96-well plates. Single clones were collected 2-3 weeks after seeding and expanded in increasingly larger cell culture plates. After successful isolation, cells were maintained with complete media containing 2 μg/mL blasticidin.

MEF cells used for the generation of iPS cells were cultured with DMEM (Invitrogen, 10569‐010) plus 10% FBS (Invitrogen, 10099141) before retroviral infection. For iPS derivation, DMEM/F12 (Invitrogen, 11330‐032) and 20% knockout serum (Invitrogen, 10828‐028) was used instead of DMEM with 15% FBS. Plat‐E cells were cultured with DMEM plus 10% FBS, 1%penicillin–streptomycin (100×) (Invitrogen, 15140‐122), 0.001 mg/ml puromycin (Merck, 540222) and 0.01 mg/ml blasticidins hydrochloride (Invitrogen, R210‐01). iPS cells were cultured on mitomycin‐C (Sigma, M0503) treated MEF cells with DMEM plus 15% FBS, 1000  U ml‐1 LIF (Millipore, ESG1107), 2 mmol/L glutamine (Sigma, G8540), 1 mmol/L sodium pyruvate (Sigma, L7900), 0.1 mmol/L β‐mercaptoethanol (Sigma, M7522) and 0.1 mmol/L non‐essential amino acids (Invitrogen, 11140‐050).

Axenic L. donovani strain 1S2D (MHOM/SD/62/1S-CL2D) clone LdBob was obtained from Steve Beverley, Washington University School of Medicine, St. Louis, MO, and cultured as described previously [30 (link)–32 (link)]. Briefly, 10⁵ logarithmic promastigotes per mL were incubated at 26°C in M199 media (Gibco) supplemented with 10% heat-inactivated FCS, 20 mM HEPES, pH 6.9, 4.1 mM NaHCO₃, 2 mM glutamine, 8 μM 6-biopterin, 10 μg/mL folic acid, 100 μM adenine, 30 μM hemin, 1x RPMI 1640 vitamins solutions (Sigma), 100 U/mL of Penicillin/Streptomycin (Pen/Step), and adjusted at pH 7.4. Axenic amastigotes were obtained by incubating 10⁶ logarithmic promastigotes per mL at 37°C and 5% CO₂ in RPMI 1640 + GlutaMAX™ -I medium (Gibco) supplemented with 20% of heat-inactivated FCS, 28 mM MES, 2 mM glutamine, 1x RPMI 1640 amino acid mix (Sigma), 1x RPMI 1640 vitamins solutions (Sigma), 10 μg/mL folic acid, 2 mM glutamine, 100 μM adenine, 100 U/mL of Pen/Step, and adjusted at pH 5.5. Relevant selective drugs were added to the medium at the following concentrations: 30 μg/mL hygromycin B (Invitrogen), 30 μg/mL puromycin dihydrochloride (Sigma), and 20 μg/mL blasticidin S hydrochloride (Invitrogen). When appropriate, axenic amastigotes cell aggregates were dispersed by passing cell suspensions five times through a 27-gauge needle before analysis.