G418 antibiotic

HepG2.2.15 cell, which has been stably transformed with two copies of the HBV genome into human hepatoblastoma cell line HepG2, was used as in vitro model to evaluate the anti-HBV effect and mechanism of IFN-CSP. The HepG2.2.15 cells were grown in complete DMEM (Gibco-BRL, CA) containing 10% FBS (Hyclone, Thermo Fisher, PA), 380 micro·g/mL G418 antibiotic (Sigma, MO), 100 units/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator with 5% CO₂ at 37°C.
IFNα2b was purchased from Beijing Kaiyin Pharmaceutical Group (Beijing, China). IFN-CSP was expressed in Escherichia coli, with a specific activity of 1.2 × 10⁸ IU/mg protein at a purity of over 95% [15 (link)].

HeLa cells were cultured in RPMI media (Gibco), supplemented with 5% fetal calf serum (FCS). HeLa.vector or HeLa.IL-36R cell lines were generated by transfection with pCXN2.empty or pCXN2.IL-1Rrp2 (IL-36R) plasmids followed by selection using G-418 antibiotic (Sigma). IL-36R over-expressing clones were expanded from single cells. Clones were selected by detection of responsiveness to active forms of IL-36 via ELISA. The HeLa.IL-36R.SEAP cell line was generated by transfection with pNifty2-SEAP plasmid (InvivoGen) followed by selection using zeocin antibiotic, as previously described^{36 (link)}. Clones were expanded from single cells and tested for SEAP production. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂.

The HeLa^IL36R cell line was generated by transfection with pCXN2.IL‐1Rrp2 (IL‐36R) plasmid followed by selection using G418 antibiotic (Sigma). IL‐36R overexpressing clones were expanded from single cells using limiting dilution cloning, followed by expansion of individual clones. The HeLa^IL36R‐SEAP cell line was generated by transfection of stable HeLa^IL36R overexpressing cells with pNifty2‐SEAP plasmid (InvivoGen) followed by selection using Zeocin antibiotic. Clones were expanded from single cells using limiting dilution cloning. Clones were selected by demonstration of acquired optimal responsiveness to active forms of IL‐36 via SEAP production and ELISA. HaCaT cells were cultured in Dulbecco's modified eagle's medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with FCS (10%). Primary neonatal foreskin‐derived keratinocytes were purchased from Cell Systems (Troisdorf, Germany) and cultured in serum‐free Dermalife K media (Cell Systems). All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂.

Purified plasmids containing the flanking regions plus the His-tagged WT and mutant coding sequences of pgk1 were used for amplification of the sequence employed for homologous recombination, using 5′F and 3′R primers (Supplementary Table 1). The corresponding PCR product (100 ng) was electroporated into 100 μl electrocompetent S. pombe prepared as described previously (Forsburg and Rhind, 2006 (link)). Yeasts were recovered in 1 M sorbitol at 30°C for 3 h, and then cells were pelleted, resuspended in 200 μl YE medium, and plated on YPD agar in the presence of 200 μg/ml G418 antibiotic (Sigma-Aldrich). Cells were grown for 4–5 days at 30°C and single colonies were picked and grown on YPD medium supplemented with 200 μg/ml G418 antibiotic. To corroborate the incorporation of synonymous mutations, the His-tagged pgk1 coding sequence (WT and mutants) were sequenced after amplification of the gDNA of 5–15 clones for each mutated region, using PGK1-F and PGK1-R primers. Additionally, to check that the recombination occurred in the endogenous pgk1 gene, we designed primers that amplify the locus segment outside the recombined segment (Supplementary Table 1), Forward_Out_PGKF and Reverse_Out_PGKR, combined with internal primers within the segment.

The LbrPGF2S CDS (coding DNA sequence) was amplified from the genomic DNA (gDNA) of L. (V.) braziliensis strain MHOM/BR/75/M2904 using primers LbrPGF2S-nostart-BglII-For (5ʹ-TCA AGA TCT GCT GGG GCC GCT GGG GCC ATC AAC GTT GGT AAG ACC G -3ʹ) and LbrPGF2S-BamHI-Rev (5ʹ-TCA GGA TCC TCA GAA CTG CGC CTC ATC A -3ʹ). The PCR products were digested with BglII and BamHI enzymes and cloned into the pmCherry-C1 (Addgene, Cambridge, MA, USA) plasmid digested with same enzymes. The mCherry-PGF2S plasmid was then digested with PmeI and NdeI enzymes. Promastigotes were transfected with mCherry-PGFS or mCherry linear fragments by electroporation [16 (link)]. Transfectant colonies were extracted from M199-agar medium in the presence of the G418 antibiotic (Sigma-Aldrich, St. Louis, MO, USA). The G418 LD₅₀ was determined for the Lb2903 strain, and at four-fold LD₅₀ drug concentration (4 µg/ml). Parasites overexpressing LbrPGF2S ectopically were produced and kept as described in [4 (link)].