Hygromycin b

Ba/F3-gp130 cells were retrovirally transduced with the pMOWS expression plasmids coding for the various IL-23R, IL-12Rβ2 and IL-12Rβ1 variants as described^{18 (link)}. Transduced cells were grown in standard DMEM medium as described above supplemented with 10 ng/ml HIL-6. Selection of transduced Ba/F3 cells was performed with puromycin (1.5 µg/ml) or hygromycin B (1 mg/ml) (Carl Roth GmbH, Karlsruhe, Germany) or both for at least two weeks. Afterwards, HIL-6 was washed away and the generated Ba/F3-gp130 cell lines were selected for HIL-23- and HIL-12-dependent growth.

Promastigotes (2 × 10⁷) in the logarithmic-growth phase were transfected with 3 to 5 μg DNA by electroporation using a human T cell Nucleofector kit (Lonza, Basel, Switzerland). After 24 h of culture in SDM, selection pressure was applied by supplementing the medium with 16 μg/ml hygromycin B (Carl Roth, Karlsruhe, Germany), 16 μg/ml G418 (Carl Roth, Karlsruhe, Germany), or 40 μg/ml bleomycin (InvivoGen, San Diego, CA, USA). After 1 week, cultures in 48-well plates were analyzed for proliferating parasites. These were passaged three times until a line was considered stable. Genomic DNA was obtained and homologous recombination was verified by PCR and sequencing. Knockout of DECR of both copies was checked and the possibility of retention of an extra copy of chromosome 33 in double knockout parasites excluded using Southern blot analyses and DECR ORF-specific PCR and Western blot analysis using a DECR-specific antiserum (see below).

Deletion of tmk1, tmk2 and tmk3 was done in QM6aΔku80 following the procedure as described previously^{80 (link)} with the hygromycin (hph) marker cassette constructed by yeast recombination of the 1 kb flanking regions up- and downstream of the gene of interest and the hph marker. Transformation was done by protoplasting and 50 µg/mL hygromycin B as selection reagent (Roth, Karlsruhe, Germany)^{115 (link)}. Protoplasts were isolated three to six days after transformation and subjected to a minimum of two rounds of single spore isolation. Successful deletion was confirmed by the absence of the gene by PCR (Table S1). All three mutants were confirmed to only have a single integration of the deletion cassette by copy number determination^{102 (link)}.

Botrytis cinerea (Pers.: Fr.) strain B05.10 was used for this study. Standard cultivation was carried out on HA medium (10 g/L malt extract, 4 g/L yeast extract, 4 g/L glucose, 15 g/L agar). B. cinerea bcrdr1 ko and bcdcl1dcl2 ko mutant strains were grown on HA medium supplemented with 70 μg/mL hygromycin B (Carl Roth GmbH). Fungal plates were incubated at room temperature in a growth chamber under long wavelength UV light (EUROLITE; 20 W to stimulate sporulation. The tomato (Solanum lycopersicum (L.) cultivar Heinz) used in this study was grown under the condition of 24°C, 16 h light/8 h dark, 60% humidity in a growth cabinet. Arabidopsis thaliana (L.) ecotype Col-0 was grown under short day condition (10 h light/ 14 h dark, 22°C, 60% relative humidity) for Botrytis infection. For H. arabidopsidis infection, Arabidopsis Col-0 was grown under long day condition (16 h light/ 8 h dark, 22°C, 60% relative humidity). Transgenic T1 and T2 A. thaliana lines were selected on 1/2 Murashige and Skoog (MS) medium supplemented with kanamycin (Carl Roth; 100 μg/mL). Independent plant transformant lines were indicated (e.g., STTM #3).

Leishmania donovani strain 1S [29 (link)] promastigotes and derived mutants were cultured at 25 °C in M199+ medium [30 (link)] with the respective antibiotics: puromycin (25 µg/mL, AppliChem, Darmstadt, Germany), blasticidin (5 µg/mL), G418 (50 µg/mL) and hygromycin B (50 µg/mL, all Carl Roth, Karlsruhe, Germany). Cells were passaged every 3–4 days.

The human pancreatic neuroendocrine cell lines Bon-1 and QGP1 were provided from T. Gress, Philipps University Marburg, Germany. Bon-1 cells were cultured in DMEM F-12 (Gibco, Invitrogen Corp. Waltham, MA, USA). QGP1 cells were grown in RPMI (Gibco, Invitrogen Corp.). Both media were supplemented with 10% FCS (Capricorn, Ebsdorfergrund, Germany). All cells were cultured in a humidified atmosphere containing 5% CO₂ at 37 °C. The cell number was analysed by counting the cells in a Neubauer chamber. The amphotropic packaging cell line LinX was maintained in DMEM, 10% foetal bovine serum, 250 mg/mL gentamicin and 100 mg/mL hygromycin B (Roth, Karlsruhe, Germany). Bon-1 cells stably expressing CUX1 were generated as previously described [22 (link)].