Calcimycin

Oseltamivir carboxylate was purchased from MedChemexpress CO., Ltd, (Monmouth Junction, NJ). Lysosomotrophic agents amodiaquin dihydrochloride dihydrate, bafilomycin A from Streptomyces griseus, chloroquine diphosphate salt, quinacrine dihydrochloride, quinidine anhydrous, Mefloquine hydrochloride, and primaquine diphosphate were purchased from Sigma-Aldrich (St-Louis, MO). Quinine sulfate was obtained from Fisher (Fair Lawn, NJ). Calcium modulators calcimycin (A23187), capsaicin, 5-(N,N-Dimethyl) amiloride hydrochloride, and verapamil hydrochoride were purchased from Sigma-Aldrich whereas TMB-8 hydrochloride was purchased from Calbiochem (Mississauga, ON). Mefloquine, calcimycin, bafilomycin A, and capsaicin were dissolved in DMSO (Sigma-Aldrich) in order to have a final solvent concentration of less than 0.1%, whereas other compounds were dissolved in culture medium. At this concentration, DMSO showed no apparent toxicity in MDCK cells (less than 1%, data not shown).

Complete media was replaced with Serum-free medium, Neurobasal-A (Gibco, Waltham, MA, USA) consisting of 1% (v/v) B-27 supplement (Gibco), one millimolar L-glutamine (Sigma), and 1% (v/v) penicillin-streptomycin (Sigma) in a total volume of 500 microliters per well in a 12-well plate. In addition to untreated controls, the following conditions were used: apoptotic inducer STS (staurosporine; 0.5 µM; Sigma) that activate calpain and caspase-3, calcium ionophore A23187 (calcimycin; 20 µM; Sigma, St. Louis, MO, USA), and maitotoxin (MTX; 10 nM; Sigma) for 16 h. A23187 and MTX both acts by activating extracellular calcium channels leading to an increase of cytosolic Ca²⁺ ions, and indirectly can activate calpain-1 and -2. The conditions mentioned above were pretreated with or without okadaic acid (OA; 100 nM; Cell Signaling, Danvers, MA, USA) for six hours prior the additions of STS, A23187, and MTX challenges.

Floating iMG were collected after 20 days of differentiation and plated on a 35 mm glass bottom dish with a coverslip (MatTek, Cat. No. NC9341562) for three days in near homeostatic iMG medium. After three days, medium was refreshed containing 2 µM Fluo4 (ThermoFisher Scientific, Cat. No F14201) and incubated for 30 min in the CO₂ cell culture incubator at 37 °C. The cells were then washed three times with Krebs-Ringer Solution (Alfa Aesar, Cat. No. J67795) containing 2.5 mM probenecid (ThermoFisher Scientific, Cat. No P36400) and were used for microscopy. To record the activity, images were acquired every 5 s on a Zeiss AXIO Observer Z1 microscope using the Zeiss ZEN software in order to record the calcium flux activity of the iMG. The activity was recorded for one minute to acquire the baseline before adding 1 µM ATP (Sigma Aldrich, Cat. No. A2383) or 0.5 mM calcimycin (positive control; Sigma Aldrich, Cat. No. C7522) following by recording for an additional five or ten minutes. The images were processed using CellProfiler software and the Fluo4 intensity values was plotted over time to obtain the activity using Prism software.

For stimulation experiments, cells were incubated with 10 µM ManICS1-AM for 2 h to allow for effective labeling and AM ester cleavage. Pharmacological stimulation was conducted by adding 5 µM thapsigargin, 25 µM carbochol, 5 µM calcimycin, or 30 µM arachidonic acid (Sigma-Aldrich, St. Louis, MO). Chemical agents were added while cells were in suspension to allow for mixing, then samples were gently pelleted the samples and collected R₁ relaxometry data.
For comparison stimulation measurements performed with Fura-2FF-AM (BioVision, Exton, PA), adherent HEK293 cells (Life Technologies) were seeded onto a 96-well plate at 5000 cells/well and grown for two days until 90% confluent. Cells were then incubated for 45 min in 5 µM Fura-2FF-AM and then washed with media. Stimulants (10 µM thapsigargin, 100 µM charbocol, 5 µM calcimycin, or 30 µM arachidonic acid) were quickly added to multiple wells via multipipettor and fluorescence output was measured at 340 and 380 nm using a Spectramax plate reader. Measurements were repeated every 5 min for 40 min to chart the time course of calcium concentration changes at room temperature. Mean responses reported in Fig. 4d were reported as fold changes in the ratio of fluorescence emission at 340 and 380 nm pre- vs. post stimulation.

Cell stress inducers were dissolved first in DMSO (maximal concentration 0.1%) and added to cells as follows: ER stress with 10 µM tunicamycin (Sigma-Aldrich, Hamburg, Germany), calcium overload with 1 μM calcimycin (Sigma-Aldrich, Hamburg, Germany). Oxidative stress with 20 µM antimycin (Sigma-Aldrich, Hamburg, Germany), autophagy induction via serum starvation (DMEM without FBS), Aβ toxicity with 1 µM Aβ 1–42 oligomers (see oligomers preparation). Also, 250 nM of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, Sigma-Aldrich, Hamburg, Germany) and 10μM of Compound W (Tocris Biosciences, Bristol, England), γ-secretase inhibitors, were used to evaluate the role of γ-secretase activity in cellular stress and MPTP opening. Furthermore, 100 μM of 2-Aminoethoxydiphenyl borate (2-APB, Sigma-Aldrich, Hamburg, Germany), an ER calcium channels inhibitor, was used to evaluate the influence of store operated calcium and 1.6 μM Cyclosporin A, was used as a MPTP inhibitor (Sigma-Aldrich, Hamburg, Germany) (see Supplementary Table 3 for mechanisms of action).

Epididymides of a single male mouse were excised and opened up in 3 ml of noncapacitating Toyoda-Yokoyama-Hosi (TYH) medium (119.37 mM NaCl, 4.78 mM KCl, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄, 5.56 mM glucose, 1.71 mM CaCl₂, and 0.5 mM Na-pyruvate), and sperms were allowed to swim out for 30 min at 37°C. Afterward, the suspension was filtered through a 100-μm cell strainer (Greiner), the total volume was adjusted to 4 ml, and the cells were counted using M-NZ Disposable Hemocytometer chips (Kisker Biotech). Afterward, cells were spun down for 5 min at 1000g for 2 min and processed further for Western blot analysis or indirect immunofluorescence. Alternatively, cells were capacitated using 25 mM NaHCO₃ (Roth) and BSA (5 mg/ml; Roth) for 45 min at 32°C, followed by induction of the acrosome reaction for an additional 45 min using 10 μM calcimycin (Sigma-Aldrich).