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AuNP conjugates were prepared using 15 nm AuNPs (1×, 1.4 × 10¹² particles mL⁻¹; BBI Solutions, Cardiff, UK) or 40 nm AuNPs (1×, 9.0 × 10¹⁰ particles mL⁻¹; BBI solutions). HRP-conjugated mouse anti-cTnI Ab (4T21C-19C7) was purchased from Hytest (Turku, Finland). The conjugate-synthesis procedures of AuNP-Ab-HRP and AuNP-(ald)HRP-Ab were described in our previous study [23 (link)]. Briefly, the AuNP-(ald)HRP-Ab conjugate (ald)HRP (Thermo Fisher Scientific, Waltham, MA, USA) was physically adsorbed to the AuNP surface, and the unbound (ald)HRP particles were eliminated via centrifugal washing. Then, an anti-cTnI Ab (4T21-19C7; Hytest) was linked to the AuNP-(ald)HRP complex through covalent coupling. For the centrifugal washing, we used centrifugation speeds of 21,000× g (for 15 nm AuNPs) and 7600× g (for 40 nm AuNPs). The AuNP-(ald)HRP-Ab conjugates were concentrated 20× in 50 μL of storage buffer containing 10 mM Fe-EDTA (Sigma), 5% (w/w) trehalose (Sigma), and 0.05% (w/w) BSA (Fitzgerald, Acton, MA, USA) in 10 mM phosphate-buffered saline (PBS; pH 7.4).
The absorbance spectra of AuNPs and AuNP-(ald)HRP-Ab conjugates were measured using a UV-2450 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). The hydrodynamic diameter was analyzed by dynamic light scattering (DLS, ELSZ-1000; Otsuka Electronics Co., Ltd., Osaka, Japan).

Wild-type Tetrahymena strains CU428.2 (mating type VII, RRID:TSC_SD00178), B2086 (mating type II, RRID:TSC_SD01627), and SB210 (mating type VI, RRID:TSC_SD00703) were obtained from the Tetrahymena Stock Center, Cornell University (https://tetrahymena.vet.cornell.edu/). ΔSPO11 (Mochizuki et al., 2008 (link)), SPO11i (Howard-Till et al., 2013 (link)), and Asf1-GFP (Garg et al., 2013 (link)) strains were constructed previously. Cells were grown at 30°C in SPP medium containing 1% proteose peptone (Becton Dickinson, Sparks, MD, USA), 0.1% yeast extract (Becton Dickinson), 0.2% glucose (Sigma-Aldrich, St. Louis, MO), and 0.003% EDTA-Fe (Sigma-Aldrich). To make them competent for mating, cells at mid-log phase (approximately 10⁶/ml) were washed with 10 mM Tris-HCl (pH 7.4), resuspended in 10 mM Tris-HCl (pH 7.4), and starved at 30°C for ~18 hr to starve. To induce mating, equal numbers of cells of two different mating types were mixed together and incubated at 30°C.

Wild-type Tetrahymena thermophila strain CU428.2 was grown at 30 °C, shaking at 100 rpm, in super proteose peptone (SPP) medium containing 1% proteose peptone (Becton Dickinson, Sparks, MD, USA), 0.1% yeast extract (Becton Dickinson), 0.2% glucose (Sigma-Aldrich, St. Louis, MO, USA), and 0.003% EDTA-Fe (Sigma-Aldrich). Cells were untreated or treated with 500 μg/mL or 2.5 mg/mL of 1,10-phenanthroline for 1, 5, or 30 min. Cells were collected, then fixed with 10% (w/v) trichloroacetic acid (TCA; Sigma-Aldrich) and incubated on ice for 30 min. After removal of TCA by centrifugation at 12,000× g for 2 min, cell pellets were lysed in PAGE sample buffer (2% SDS ([Sigma-Aldrich], 2.5% 2-mercaptoethanol [Sigma-Aldrich], 10% glycerol [Sigma-Aldrich], and 50 mM Tris-HCl, pH 6.8) and boiled for 5 min prior to analysis by SDS-PAGE and immunoblot. Antibodies used were anti-Tetrahymena SUMO (1:1000; Gift from Jim Forney), or anti-β-actin antibody (1:1000; GenScript, Piscataway, NJ, RRID:AB_914102).