Cyanine3 (cy3)

Plasma samples from HF and HDF patients (15 μg of protein) were separated and analysed by denaturating sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) of 12% acrylamide (3% cross-linking). Low molecular weight calibration kit (GE Healthcare) for SDS-PAGE was used as molecular mass standards.
In DIGE experiments, plasma samples from HF and HDF patients (5 μg of protein) were labelled with Cy3 or Cy5 fluorescent dyes (GE Healthcare) according to the standard protocol for DIGE assay, as previously described^{21 (link)}. Labelling was performed in samples from HF and HDF patients (n = 9 per group) in a paired combination. A sample of HF (patient 1) was randomly discarded and the labelling was alternated between Cy3 and Cy5 in each group. After fluorescence labelling, HF and HDF samples were combined and analysed by SDS-PAGE as described above. In the Cy-labelled experiments for subsequent MALDI-TOF MS identification pools (n = 5) of both HF and HDF patients’ plasma samples were combined. A detailed protocol is included in the Supplementary Material.
Coomassie brilliant blue (R-250, BioRad) was used to stain the proteins in gels, that after were washed-out with 20% ethanol/7% acetic acid (vol/vol). Finally, gels were preserved in 10% ethanol until scanning and protein quantification, or extraction for subsequent identification by MS.

MFN1_IM(C156S + A696C), MFN2_IM(C390S + A706C), and related mutants were labeled with fivefold concentration of fluorescent dye Cy3 or Cy5 (GE Healthcare) for 60 min at 4 °C in a buffer containing (20 mM HEPES, pH 7.5, 150 mM KCl, 5 mM MgCl₂, and 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP). Free dye was removed by gel filtration chromatography using a Superdex 200 10/30 column. Cy3 was excited at 537 nm, with peak emission at 570 nm. Cy5 fluorescence emission was detected at 667 nm. Cy3- and Cy5-labeled proteins (1 μΜ each) were mixed in a buffer containing 20 mM HEPES, pH 7.5, 150 mM KCl, 5 mM MgCl₂, and 2% β-ME as previously described^{33 (link)}. Fluorescence was measured once every 1 min in a flat black 96-well plate for 40 min, then 2 mM indicated nucleotide was added and the measurement continued for another 60 min. FRET traces were calculated as: FRET = I_Cy5/(I_Cy3 + I_Cy5), where I_Cy3 and I_Cy5 are the instantaneous Cy3 and Cy5 fluorescence intensities, respectively.

DNA microarrays (60-mer, Agilent Technologies, USA) based on the full P. falciparum genome as previously described [96 (link)] were used to assess global transcriptomic changes in gametocytes treated with JIB-04. Day 2 and day 3 gametocyte cultures (1–3% gametocytaemia, 4% haematocrit) were treated with 5 µM JIB-04 (Cayman Chemicals) for 24 h followed by isolation of gametocytes using 0.01% (w/v) saponin. Total RNA was isolated with a combination of TRIzol (Sigma-Aldrich, USA) and phenol–chloroform extraction and subsequently used to synthesise cDNA as previously described [96 (link)] for the untreated and JIB-04 treated day 2 and day 3 gametocyte samples. Sample cDNA was labelled with Cy5 dye (GE Healthcare, USA) prior to hybridisation to arrays with an equal amount (350–500 ng) of Cy3-labelled (GE Healthcare, USA) reference pool containing equal amounts of cDNA from each gametocyte sample and mixed stage 3D7 asexual parasites. After hybridisation, the slides were scanned on a G2600D (Agilent Technologies, USA) scanner and normalised signal intensities for each oligo were extracted using the GE2_1100_Jul11_no_spikein protocol and Agilent Feature Extractor Software (v 11.5.1.1) as described before [96 (link)].

PBMC protein samples were labeled with fluorescent cyanine (Cy) dyes (GE Healthcare, Singapore). Six FRDA patients and age- and gender-matched healthy controls were selected for proteomics analysis (six biological replicates). The internal standard was prepared by combining equal amounts of each of the 12 samples. Prior to fluorescent dye labeling, the pH of each sample was adjusted to around 8. For each sample, 50 μg of proteins was labeled with 200 pmol of either Cy3 or Cy5, using minimal labeling method (GE Healthcare). Dye swapping was done to ensure no biasing of Cy dyes for any protein sample. Internal standard was labeled with 200 pmol of the Cy2 fluorophore in each experimental set (Table 1). The labeling was performed in the dark and on ice for 30 min. The reaction was stopped by adding 10 mM lysine and the samples were stored on ice for 15 min. Afterward, the Cy2, Cy3, and Cy5 sample were pooled in a single tube.