Gene pulser 2 system

To generate stable transfectants expressing Zap70^wt or Zap70^C564A, P116 cells were transfected with 5–30 µg of pPB[Exp]-Puro-EF1A>hZAP70/T2A/EGFP and 5 μg of pEF_hyPBase. For transient transfections of P116 cells, we used 5–30 µg of the pEYFP-N1-hZap70 vector. DNA electroporation of P116 cells was performed using the Gene Pulser II System (BIORAD) as previously described [16 (link)]. The transfected cells were cultured in an RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. A total of 0.5 μg/mL of puromycin (Gibco, Waltham, MA, USA) was added for the generation of stable transfectants, which were additionally sorted with the Aria Cell Sorter 3 (BD Bioscience) and afterwards maintained in RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1% Ciprobay.
For the transfection of HEK293T cells, 1 × 10⁶ cells were seeded onto 6-well tissue culture plates one day before transfection. Separately, a mixture was prepared containing 300 μL of 250 mM CaCl₂ and 10 μg of pEYFP-N1-hZap70, which was added dropwise under constant agitation to 300 μL of HEPES buffered saline (Sigma-Aldrich). After incubation for 45 min at RT, 3 mL of DMEM (PAN Biotech, Aidenbach, Germany) were added to the mixture, which was subsequently carefully pipetted into the well containing the cells. Cells were incubated in the presence of a transfection medium for 24 h.

Plasmid DNA was isolated from overnight E. coli cultures using an alkaline lysis method (modified from Ish-Horowicz and Burke, 1981 (link); Sambrook and Russell, 2001 ) and transformed into electrocompetent E. coli (ElectroMax DH5α, Invitrogen), or electrocompetent A. tumefaciens (Wise et al., 2006 (link)), using the Gene Pulser II System (Bio-Rad) set to 2.0 kV, 25 µF capacitance, and 200 Ω or 400 Ω resistance, respectively. Immediately following electroporation, E. coli and A. tumefaciens cells were incubated for 1 h in non-selective LB medium, before plating on selective medium. Correct insertion of amplicons into plasmid DNA of positive E. coli transformants was confirmed by restriction enzyme digestion and sequencing of isolated plasmid DNA. pCB-ADTs were transformed into A. tumefaciens LBA4404 containing Ti-helper plasmid pAL4404 (Hoekema et al., 1983 ; Hellens et al., 2000 (link)).

Electrocompetent cells of E. coli were prepared as described previously (38 ). For Burkholderia strains requiring induction of gene expression from the araB promoter, L-arabinose was added at a final concentration of 2% when cells grown in LB medium reached an OD_640nm of 0.8–1.0 and further grown for 3 hours before preparing them as electrocompetent cells. For electrocompetent cells’ preparation, a volume of 100 mL of culture was transferred to a centrifuge tube and cooled in ice for 15 minutes, followed by centrifugation at 5,000 × g for 5 minutes at 4°C. The supernatant was removed, and the cell pellet was washed three times with 40 mL ice-cold distilled sterile H₂O, followed by two washing steps with 20 mL of 10% ice-cold glycerol. In the end, the cell pellet was resuspended in 1 mL of 10% glycerol, and 50 µL aliquots were immediately frozen at −80°C. Electroporation was performed using the BioRad Gene Pulser II system with the following parameters: 25 μF, 2.5 kV, and 200 Ω for Burkholderia and 25 μF, 2.5 kV, and 400 Ω for E. coli. Cells were recovered in the LB medium for 1 hour (E. coli) or 4 hours (Burkholderia) before being plated in a selective medium. Triparental conjugation was performed as described previously (40 (link)) using plasmids pRK600 or pRK2013 as helper plasmids.

pfhsp70-1-mCherry was subcloned into a P. falciparum expression vector, pfYC103 as previously reported (Wagner et al., 2013 (link)). The plasmid was purified using a Qiagen Maxi kit and resuspended in CytoMix (25 mM HEPES, pH 7.6, 2 mM EGTA (Sigma), 5 mM MgCl₂ (Fisher Scientific), 8.66 mM K₂HPO₄ (Fisher Scientific), 1.34 mM KH₂PO₄ (VWR International), 120 mM KCl (Fisher Scientific), 0.15 mM CaCl₂ (Sigma)) (Rug and Maier, 2013 (link); Crabb et al., 2004 (link)). For transfection of P. falciparum 3D7, 100 μL ring-stage parasites (14–18 hpi) at 5% parasitemia was resuspended in 300 μL CytoMix containing 75 μg plasmid DNA in a 0.2 cm electroporation cuvette (Bio-Rad). Electroporation was performed at 0.31 kV and 950 μF with maximal capacitance using a Gene Pulser II system (Bio-Rad). The parasites were then cultured in complete medium at 1% hematocrit at 37°C. Selection of transfectants with 200 nM pyrimethamine (Sigma) started at 3 days post-transfection. PfHsp70-1-mCherry expression in transfected parasites was verified by fluorescence microscopy.

Sub-confluent (60–80%) HUVECs primary cultures were resuspended in serum-free M199. Aliquots of the cell suspension (0.5 mL, 3.2 x10⁶ cells/mL) were mixed with 10 μg of the pGL3-hCAT1^-1606 or pGL3-hCAT1^-650 constructs, pGL3-Basic (empty pGL3 vector), pGL3-Control (Simian Virus 40 promoter (SV40) pGL3 vector), and the internal transfection control vector, pRL-TK expressing Renilla luciferase (Promega) [19 (link), 20 (link)]. After electroporation (300 Volts, 700 μF, 5–10 milliseconds) (Gene Pulser II System, BioRad, CA, USA), the cells were cultured (49 hours) in M199 containing 2% FCS. The transfection efficiency was estimated by transfection of the pEGFP-N3 vector (Clontech, Mountain View, CA, USA), and the fluorescent cells were counted under an inverted fluorescent microscope (Leica DMIL; Wetzlar, Germany) [19 (link), 20 (link)].

Resuspended exosomes were diluted in the Gene Pulser electroporation buffer (Bio-Rad Laboratories, CA) in 1 : 1 ratio. 1 μmol of mouse miR-132 mimic (Ambion, NY) or inhibitor was added to 200 μl of exosome sample. The mixtures were transferred into cold 0.2 cm electroporation cuvettes and electroporated at 150 V/100 μF capacitance using a Gene Pulser II system (Bio-Rad Laboratories, CA) as described previously [18 (link)]. After removing the free-floating miRNA mimic, exosomes were reisolated using ultracentrifugation. The final pellet (exosome) was resuspended in PBS and stored at −80°C.