Journal: bioRxiv

Article Title: Doxycycline inhibits both apicoplast and mitochondrial translation in apicomplexan parasites

doi: 10.64898/2026.03.11.710986

Figure Lengend Snippet: A) Seahorse metabolic flux assays were used to measure the activity of the electron transport chain (ETC) through determining the oxygen consumption rate (OCR). ETC complex inhibitors such as atovaquone (Atov) and NaN₃ decrease the OCR while electron donors such as malate and TMPD, reduced by ascorbate (Asc), can deliver electrons directly to ubiquinone (Q) and cytochrome c (Cyt c ) respectively. B and C) Example trace of Plasmodium falciparum treated with doxycycline (DOX, blue triangles), which has severely reduced basal mOCR (initial malate-dependent OCR minus OCR after Atov injection) and TMPD-dependent mOCR (OCR following TMPD injection minus OCR after Atov injection) compared to the DMSO control (grey circles). Data presented as mean ± SD (n = 3 biological replicates (2-3 technical replicates per condition)). Unpaired t-test, p <0.05 *, <0.01 **, <0.001 ***. D) OCR measurements of Toxoplasma gondii permeabilised parasites in the presence of either DMSO, doxycycline or clindamycin (CLI, green squares) for 1-2 days. Data presented as mean ± SD (n = 3) E) Basal malate-elicited and TMPD-elicited mOCR of parasites grown in the presence of DMSO, DOX, or CLI for 1–2 days. Data depict the mean ± 95% CI of the linear mixed-effects model (n = 3). ANOVA followed by Tukey’s multiple pairwise comparisons test was performed: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. F) T. gondii D ku 80/TATi tachyzoites were treated for 24 h with 1, 25 or 50 µM DOX, or 0.002 or 10 µM CLI. Whole cells we solubilised in 2% (w/v) DDM and the soluble material loaded for clear or blue native PAGE analysis of mitochondrial protein complexes. Complex IV activity was analysed using an in-gel cytochrome c : 3,3’- diaminobenzidine (DAB) stain, and complex V assembly by Western blot (representative plot shown), probing with rabbit anti-Atpβ. G-J ) Western blots of proteins extracted from Tg Cox2a-HA/ Tg QCR11-FLAG parasites cultured in the presence of DMSO, 25 µM DOX or 10 µM CLI for 1-2 days, separated by ( G-H ) BN-PAGE or ( I-J ) SDS-PAGE, and probed with anti-FLAG antibodies to detect the complex III protein Tg QCR11-FLAG, anti-HA antibodies to detect the complex IV protein Tg Cox2a-HA, or anti- Tg Tom40 antibodies as a loading control. Data are representative of two independent experiments.

Article Snippet: For the western blotting, membranes were probed with rat anti-HA (1:2,000 dilution; Merck clone 3F10, catalogue number 11867423001), mouse anti-FLAG (1:1,500 dilution; Merck clone M2, catalogue number F3165), or rabbit anti- Tg Tom40 (1:2,000 dilution, ( )) primary antibodies, and horseradish peroxidase-conjugated goat anti-rat (1:10,000 dilution; Abcam catalogue number ab97057), goat anti-mouse (1:10,000 dilution; Abcam catalogue number ab6789) or goat anti-rabbit (1:10,000 dilution; Abcam catalogue number ab97051) secondary antibodies.

Techniques: Activity Assay, Injection, Control, Blue Native PAGE, Staining, Western Blot, Cell Culture, SDS Page