Cytomix

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.

Wound Healing Assay was assessed by scratch test as previously described (19 (link)). HT29 cells were cultured in 6-well plated at a density of 2 × 10⁵ cells/ml until confluence reached 90%. A straight-line wound was made using a 10-ul pipette tip. Cell debris and smoothed the edge of the straight-line wound were removed by a wash with PBS and cells were then maintained in a medium with a reduced percentage of FBS (1%). Then cells were exposed to cytomix [TNFα (100 ng/ml) and INFγ (250 ng/ml), Peprotech, Rocky Hill, USA] or to B-box (10 μg/ml) (HMGBiotech, Milan, Italy) in presence or absence of DPG (300 μM) or anti-HMGB1 antibody (Sigma) for 48 h. In a second set of experiments, cells were treated with cytomix for 24 h and then were exposed to DPG (300 μM, Sigma), anti-VTN (1:1,000) and anti-PLAUR (1:1,000), alone or in combination, for 24 h. In both case, cells migrated into the wounded area were visualized at 0, 6, 24, and 48 h by Hematoxylin and Eosin staining. Images of each condition were acquired at a magnification of 10X. Cellular density related to a fixed wounded area (1 mm²) was measured after 48 h using ImageJ software (available in the public domain at www.nih.gov; National Institutes of Health [NIH], Bethesda, MD, USA) (10 acquisition for each experimental point). The experiment was replicated three times.