Reticulocytes

Sexual differentiation was induced in tightly synchronized P. falciparum parasites (28±2 hpi) by incubating the cells for 22 hours in CM (220 μL per well of a 96-well plate; 0.3-0.5% parasitemia; 2.5% hematocrit) or in −SerM as described (Brancucci et al., 2015 (link)). If not stated otherwise, cell line Pf2004/164tdTom was used for all experiments. To determine the effect of culture perturbations on sexual commitment, serum fractions as well as nutrients or inhibitor compounds (solved in either RPMI, DMSO, chloroform, ethanol or methanol) were added to the bottom of empty wells (glass bottom dishes were used for chloroform-containing samples) and directly resuspended in parasite culture after allowing volatile solvents to evaporate. To determine sexual differentiation in reticulocyte-enriched blood, tightly synchronized parasites were magnet purified at 46±2 hpi using MACS CS columns in a SuperMACS (Miltenyi Biotec) before incubating pure schizont-infected erythrocytes (>99%) with the blood sample to be tested. These culture perturbations were then tested for effects on parasite sexual differentiation as described (Brancucci et al., 2015 (link)). In brief, following the 22 hour testing phase (see above), cells of each well were washed 3 times in 200 μL +SerM medium before being resuspended in 220 μL +SerM medium. Henceforth, medium was exchanged daily. Parasitemia and gametocytemia was quantified using flow cytometry at 20-30 hpi (MACS Quant, Analyzer 10) and 72-96 hpi (BD Fortessa), respectively. Cytometry data were analysed using FlowJo software and sexual differentiation rates were determined by dividing gametocytemia of each well with the corresponding parasitemia measurements. Assays were run in biological triplicates. Each biological replicate contained technical triplicates.
P. berghei sexual commitment assays were performed using a parasite line expressing an RFP reporter under the gametocyte-specific gene PBANKA_1018700 (Sinha et al., 2014 (link)) and GFP under the constitutive PBANKA_0905600 promoter, in the 507cl1 background line (RMgm-7). Mature schizonts were intravenously (IV) administered to naïve TO mice. Ring stage parasites were isolated at 4 hpi and mature trophozoites and gametocytes were removed by passing through a MACS LD column (Miltenyi Biotec). Infected erythrocytes were incubated in −SerM medium, −SerM medium supplemented with 20 μM LysoPC (−SerM/LysoPC), or serum-complemented medium (+SerM) for 20 hours. Mature schizont stage parasites were then isolated on a 55% Nycodenz (Axis-Shield POC)/RPMI gradient and injected intravenously into 2 or 3 naïve mice. GFP-expressing cells were examined by flow cytometry at 16 hpi to calculate parasitemia, while cells expressing both RFP and GFP (gametocytes) were assessed at 21 hpi. Gametocytemia was calculated as [(RFP⁺ and GFP⁺ cells)/GFP⁺ cells]^∗100.

Recombinant GST-N-Myc protein was incubated with or without recombinant p300 protein in a ubiquitination reaction containing 20 mM Hepes (pH 7.5, catalog no.: H3375; Sigma–Aldrich), 5 mM MgCl₂ (catalog no.: AM9530G; Thermo Fisher Scientific), 2 mM dithiothreitol (catalog no.: R0861; Thermo Fisher Scientific), 2 mM ATP (catalog no.: A6559; Sigma–Aldrich), 5 mg of ubiquitin (catalog no.: U5507; Sigma–Aldrich), 20 mM MG132, and 5 μl/reaction crude rabbit reticulocyte lysate (catalog no.: L4151; Promega) for 1 h at 30 °C (24 (link)). Products were then subjected to IP using GST antibody (catalog no.: 2624; Cell Signaling Technology) and protein A/G magnetic beads before Western blot.

Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific. RNAs were column-purified in accordance to the manufacturer’s instructions (RNeasy Mini Kit, QIAGEN), and efficient poly(A) tailing was evaluated by agarose gel electrophoresis. CREB3L2 and ATF4 translation reactions were incubated separately for 90 min at 30°C following vendor’s guidelines (Rabbit Reticulocyte Lysate System, Promega). CREB3L2 and ATF4 protein products were confirmed by immunoblotting in pilot experiments. Upon completion of the translation protocol, lysates (45 μl) containing CREB3L2 and ATF4 were mixed and incubated at 37°C for 30 min with gentle agitation (300 rpm for 5 s every minute) before immunoprecipitation, as previously described (71 (link)). Immunoprecipitation was carried out overnight at 4°C with gentle rotation in PBS supplemented with 0.1% NP-40 and protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail, Roche), using magnetic beads conjugated with anti-HA or anti-V5 antibodies (anti-HA beads: PI88836, Thermo Fisher Scientific; anti-V5 beads: NC0777490, MBL International). Washing cycles were repeated five times with immunoprecipitation buffer. Complex elution was performed in 2× Laemmli buffer [130 mM tris-Cl (pH 6.8), 0.1 mM dithiothreitol, 20% (v/v) glycerol, and 4% SDS diluted in water] by boiling at 95°C for 5 min. Immunoblot detection of tagged CREB3L2 and ATF4: anti-HA (1:4000; ab9110, Abcam), anti-V5 (R960-25, Thermo Fisher Scientific), and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000; 60004-1-Ig, Proteintech), in conjugation with TrueBlot anti-rabbit IgG horseradish peroxidase (HRP) (1:1000; Rockland) or Superclonal anti-mouse IgG HRP (1:10,000; A28177, Thermo Fisher Scientific).