Fetal bovine serum (fbs)

For cell culture assays, A549 cells and primary fibroblasts were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1% gentamicin (Sigma-Aldrich, St. Louis, MO, USA). Cultures were incubated under standard conditions at 37 °C in a humidified atmosphere containing 5% CO₂. The culture medium was replaced every three days to ensure optimal nutrient availability and to remove cellular waste. Cells were passaged when they reached approximately 80% confluence using 0.05% trypsin-EDTA (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for detachment. Experiments were initiated when cells reached the required confluence, depending on the specific assay.

All cells were grown at 37°C in the presence of 5% CO₂. Parental Flp-In T-Rex DLD-1 osTIR1 and DLD-1^YFP-AID cells were a kind gift from D. C. Cleveland (University of California, San Diego, CA, USA). DLD-1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech), and parental Flp-In T-Rex hTERT RPE-1 cells were a kind gift from J. Pines (Institute of Cancer Research: London, UK) and hTERT RPE-1 cells expressing endogenously tagged CENP-U-FKBP^F36V were grown in DMEM/F12 media (PAN Biotech); all media were supplemented with 10% tetracycline-free fetal bovine serum (Sigma-Aldrich) and L-glutamine (PAN Biotech). K562-SSRP1-dtag cells were a kind gift from P. Cramer, M. Oudelaar, and K. Žumer (Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany) and were grown in RPMI (Gibco) media supplemented with 1× GlutaMAX (Gibco).

Vero MARU (VM; Middle America Research Unit, Corozal, Panama), Vero (ATCC CCL-81, Manassas, VA, USA), and MRC-5 cells (ATCC^®CCL-171™, Manassas, VA, USA) were cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, New York, NY USA), supplemented with 5 or 10% fetal bovine serum (R&D Systems, Minneapolis, MN, USA; Atlas Biologicals, Fort Collins, CO, USA) and 1% antibiotic-antimycotic solution (ThermoFisher Scientific, Waltham, MA, Waltham, MA, USA) at 37 °C with 5% CO₂ atmosphere in a cell culture incubator. Culex tarsalis (CxTxR2; generated at ABADRU, USDA, Manhattan, KS, USA) cells and Aedes albopictus C6/36 cells (ATCC^® CRL-1660™, Manassas, VA, USA) were maintained in Schneider’s Drosophila medium (ThermoFisher Scientific, Waltham, MA, USA) and Leibovitz-15 medium (ATCC-30-2008™, Manassas, VA, USA), respectively, supplemented with 10% tryptose phosphate broth (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (IFBS, Sigma-Aldrich, St. Louis, MO, USA), and 1% antibiotic- antimycotic solution. Additionally, the CT (Cx. tarsalis) embryonic mosquito cell line was maintained in Schnieder’s Drosophila medium supplemented with sodium bicarbonate (Gibco, ThermoFisher Scientific, Waltham, MA, USA), L-Glutamine (Sigma-Aldrich, St. Louis, MO, USA), 20% heat-inactivated FBS (Atlas Biologicals, Fort Collins, CO, USA), and 1% PenStrep (ThermoFisher Scientific, Waltham, MA, USA) [21 ].
Rift Valley fever phlebovirus strains Kenya-128B-15 (Ken06; GenBank: KX096938, KX096939, and KX096940), SA01-1322 (SA01; Genbank KX096941, KX096942, and KX096943), and ZH501 (GenBank: DQ380149, DQ380200, and DQ375406) strains were amplified in C6/36 cells or Vero cells [22 (link),23 (link),24 (link)]. The live attenuated strain MP-12 strain (Genbank: DQ375404, DQ380208, and DQ380154) was propagated in MRC-5 for in vitro or Vero cells for in vivo infection [25 (link),26 (link)]. All virus-containing materials (cell culture supernatants, and tissue homogenates) were titrated by a standard plaque assay as described previously and below for each assay [27 (link)].

Because of the clonal instability of the PC12 cell line (Fujita et al. 1989 (link)), the experiments were performed on cells that had undergone fewer than five passages, and all studies were repeated several times with different batches of cells. As described previously (Crumpton et al. 2000a (link); Qiao et al. 2003 (link); Song et al. 1998 (link)), PC12 cells (1721-CRL; American Type Culture Collection, Manassas, VA) were seeded onto 100-mm poly-d-lysine-coated plates in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% inactivated horse serum (Sigma Chemical Co., St. Louis, MO), 5% fetal bovine serum (Sigma Chemical Co.), and 50 μg/mL penicillin streptomycin (Invitrogen). Cells were incubated with 7.5% CO₂ at 37°C, and the medium was changed every 2 days. For studies in the undifferentiated state, cells were seeded at varying densities so that, regardless of the total time of incubation, the cells would reach a final confluence of 60–70%. Twenty-four hours after seeding, the medium was changed to include the various test substances: chlorpyrifos (Chem Service, West Chester, PA), diazinon (Chem Service), parathion (Chem Service), physostigmine (Sigma Chemical Co.), dieldrin (Chem Service), or NiCl₂ (Sigma Chemical Co.). Because of their poor water solubility, the pesticides were dissolved in dimethyl sulfoxide (Sigma Chemical Co.), achieving a final concentration of 0.1% in the culture medium; accordingly, all cultures included this vehicle, which had no effect on the PC12 cells (Qiao et al. 2001 (link), 2003 (link); Song et al. 1998 (link)).
For studies in differentiating cells, 3 × 106 cells were seeded; 24 hr later, the medium was changed to include 50 ng/mL 2.5 S murine NGF (Invitrogen), and each culture was examined under a microscope to verify the subsequent outgrowth of neurites. The test agents were added concurrently with the start of NGF treatment.

As described previously (Crumpton et al. 2000 (link); Qiao et al. 2003 (link); Song et al. 1998 ), PC12 cells (1721-CRL; American Type Culture Collection, Rockville, MD) were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% inactivated horse serum (Sigma Chemical Co., St. Louis, MO), 5% fetal bovine serum (Sigma), and 50 μg/mL penicillin–streptomycin (Invitrogen); cells were incubated with 7.5% CO₂ at 37°C, and the medium was changed every 2 days. Because of the clonal instability of the PC12 cell line (Fujita et al. 1989 (link)), the experiments were performed on cells that had undergone fewer than five passages, and studies were repeated several times with different batches of cells. For studies in the undifferentiated state, 3–6 × 10⁶ cells were seeded onto 100-mm poly-l-lysine–coated plates, and 24 hr later the medium was changed to include 5 or 50 μM CPF (purity, 98%; Chem Service, West Chester, PA). CPF was dissolved in dimethyl sulfoxide (DMSO), achieving final DMSO concentrations of 0.1–1% in the culture medium, and the corresponding control samples contained equivalent DMSO concentrations. Preliminary studies were conducted to verify that these concentrations of DMSO had no effect on the measured parameters in PC12 cells (data not shown), in agreement with earlier work (Qiao et al. 2001 (link), 2003 (link); Song et al. 1998 ).
For studies during differentiation, 3.5 × 10⁶ cells were seeded; 24 hr later the medium was changed to include 50 ng/mL NGF (Sigma), and over the ensuing week, each culture was examined under a microscope to verify the outgrowth of neurites. CPF or physostigmine (Sigma) was added either simultaneously with the addition of NGF or after a 4-day NGF pretreatment.

Experiments were conducted in either SH-SY5Y or ARPE-19 cells following American Type Culture collection (ATCC) guidance. In brief, cells were cultured in 1:1 DMEM:F-12 media (Thermo Scientific; #12634010) supplemented with 10 % (v/v) FBS (Sigma; #F7524), 2 mM l-glutamine (Thermo Scientific; #25030-024), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Thermo Scientific; #15140-122) and incubated at 37 °C with 5 % CO₂ in a water-saturated incubator. The mito-QC reporter was retrovirally transfected as previously described (Allen et al., 2013 (link)). Mitophagy was assessed from pools of cells transfected with the reporter. Iron chelation induced mitophagy with Deferiprone (DFP) (Sigma; #379409) was achieved as previously reported, where cells were treated with 1 mM DFP, dissolved in water before use with a pulse of heat (90 °C), for 24 h. For hypoxia treatment cells were transferred to a Ruskinn INVIVO2 300 workstation (Ruskinn/Baker) at 37 °C in 1 % O₂ and 5 % CO₂, where were incubated for 42 h. After 36 h incubation in hypoxia, 50 nM of Bafilomycin A1 (Enzo; BML-CM110) was added to inhibit lysosome acidification during the remaining 6 h up to 42 h.
The experiments using the mito-QC reporter to assess mitophagy were processed as previously described (Allen et al., 2013 (link)). In brief, cells containing the mito-QC reporter were washed once with PBS (Thermo Scientific; #14190-094) and fixed with 3.7 % PFA (Sigma; P6148) in 200 mM HEPES (Formedium; HEPES10), pH 7.0 for 10 min at room temperature. Then, samples were washed twice with DMEM media supplemented with 10 mM HEPES pH 7.0 and 0.04 % Sodium Azide (Sigma; S8032), followed by a 10 min incubation. After two washes with PBS, coverslips were incubated with 1 μg/ml Hoechst (Thermo Scientific; #62249) for 30 min, washed three times with PBS, dipped in MilliQ water and mounted onto glass slides with ProLong Diamond Antifade Mountant (Thermo Scientific; #P36961).
The immunostaining of SH-SY5Y mito-QC reporter cells with LAMP1 was performed as follow: cells were washing once with PBS and fixing cells with 3.7 % PFA pH 7.0 for 20 min at room temperature. The cross-linking reaction was quenched as described above. Cells were the permeabilised with 1 % BSA (Roche; #10735108001)/PBS + 0.1 % NP-40 (Merck/Millipore; #492016) for 3 min at room temperature. In contrast to tissue preparations, the permeabilization of cell in vitro can affect the red-only mitolysosome stability. Therefore, the quantification of mitophagy using the mito-QC reporter should be avoided after detergent permeabilization of cells in vitro. Following permeabilization and fixation, coverslips were next washed two times with 1 % BSA/PBS and followed by a 30 min incubation at room temperature. The LAMP1 primary antibody (Santa Cruz; sc-20011) were incubated at 1/300 in 1 % BSA/PBS for 1.5 h at 37 °C. Then, coverslips were washed with 1 % BSA/PBS four times of 10 min each. The secondary anti-mouse Alexa Fluor 647 nm antibody (Thermo Scientific; #A21236) was incubated at 1/1000 in 1 % BSA/PBS for 30 min in the dark at room temperature. Coverslips were washed again four times with 1 % BSA/PBS for 10 min each. Cells were then stained with Hoechst and mounted glass slides as described above.
Images with the mito-QC reporter to quantify mitophagy were acquired using a Nikon Eclipse Ti widefield microscope (Plan Apo Lambda 60x Oil Ph3 DM) with the NIS-Elements software, while the images for the LAMP1 co-localisation experiment were acquired in a Leica SP8 laser scanning confocal microscope (HC PL APO 63x/1.40 oil CS2). All the images were processed with FIJI v1.52n software (ImageJ, NIH). Quantification of mitophagy was performed from three independent experiments counting over 50 cells for condition. Images were processed with the mito-QC Counter. For images acquired in the widefield microscope the following parameters were used: Radius for smoothing images = 1, Ratio threshold = 0.6, and Red channel threshold = mean + 0 standard deviation. For images acquired in the Leica SP8 confocal microscope the following parameters were used: Radius for smoothing images = 2, Ratio threshold = 1.5, and Red channel threshold = mean + 0.5 standard deviation.