RTgill-W1 cell line was obtained from American Type Culture Collection (ATCC) and grown in Leibowitz’s L-15 Glutamax (Gibco, Thermo Fisher, Waltman, MA, USA) supplemented with 10% FBS (EU standard, Biosera, Nuaille, France) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland). The cells were cultured at 19 °C in a non-ventilated cell culture flask and sub-cultured 1:2 once every 10th day using trypLE (Gibco). For experiments, the cells were seeded (132,000/cm2) 2 days before the experiments. The cell number gave full confluence at the day of experiment. The ASG-10 cell line was previously made by our lab (Gjessing et al. 2018) and grown in Leibowitz’s L-15 Glutamax medium supplemented with 10% FBS, 1% penicillin/streptomycin and 30 µM β-merceptaethanol (Sigma-Aldrich, St-Louis, MO, USA). The cells were cultured at 19 °C in a non-ventilated cell culture flask and sub-cultured 1:2 every 10th day using trypLE. For experiments, the cells were plated out as described for RTgill-W1, using cell culture medium without β-merceptaethanol. Rotenone (Sigma-Aldrich) was dissolved in DMSO and stock solutions (0.05, 0.5 and 5 mM) stored at − 20 °C. Unpon exposure, the medium was replaced with exposure medium (L-15 with 10% FBS and 1% penicillin/streptomycin) with or without rotenone and inhibitors as indicated. The final concentration of DMSO in the cell culture was 0.1%. Appropriate controls containing the same amount of solvent were included in each experiment. N-actyl-l-cysteine (NAC; Sigma-Aldrich) and l-Buthionine-S,R-sulfoximine (BSO; Bio-techne, Minneapolis, MI, USA) were prepared fresh in front of each experiment and dissolved in exposure medium as described above. The NAC solution was pH adjusted to 7.4.

Metabolic activity/ viability of the cells was measured using the Alamar Blue assay according to the manufacturer’s protocol (Invitrogen, Thermo Fisher). The dark blue oxidized form of Alamar Blue (resazurin) is reduced to a highly fluorescent form (resorufin) by mitochondrial or cytoplasmatic enzymes (Rampersad 2012). The measured fluorescence intensity is thus proportional to the number of viable cells. The fluorescence; Excitation (Ex) 555 nm/Emission (Em) 585 nm was quantified using Spectramax i3x plate reader (Molecular Devices, San Jose, CA, USA).

CellTox green is a non-toxic dye that stains DNA of cells with impaired membrane integrity. The binding with DNA produces a fluorescence signal that is proportional with cytotoxicity (necrotic and late apoptotic cells). CellToxTM Green Dye (1:2000; Promega, Madison, WI, USA) was added to the cells as described by the manufacturer and fluorescence (Ex 485 nm/Em 520 nm) quantified by Spectramax i3x plate reader. To ensure a representative readout of the fluorescent adherent cells, 37 different points were read in each well using the well scan function of the plate reader.

The cells were seeded on microscopy polymer coverslips (Ibidi, Gräfelfing, Germany) and treated with rotenone (0.5 µM, 2 h). The cells were stained with MitoTracker Red CMXRos (Molecular Probes, Invitrogen; 40 nM) in L-15 culture medium for 20 min at 19 °C. The staining solution was replaced with phenol red free L-15 medium with 10% FBS and 1% penicillin/streptomycin and mitochondria visualized by confocal microscopy (Zeiss LSM710, 63 × NA 1.4 oil objective).

Mitochondrial membrane potential was determined using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1; Biotium, Fremont, CA, USA), which accumulates and aggregates in intact mitochondria, emitting bright red fluorescence (Ex 497 nm/Em 595 nm). With disruption of the mitochondrial membrane potential, these aggregates do not form, and JC-1 remains in its monomeric form emitting a green fluorescence (Ex497/Em528). Microscopic analysis: The cells were seeded on microscopy polymer coverslips (Ibidi) and treated with rotenone (0.5 µM, 2 h). The cells were stained with JC-1 according to the protocol for 20 min at 19 °C. The staining solution was removed then and phenol red free L-15 medium were added before visualized by confocal microscopy (Zeiss LSM710, 63 × NA 1.4 oil objective, using Ex 488 nm/Em 493–582 nm (green) and Ex 488 nm/Em 599–758 nm (red). Flow cytometric analysis: Rotenone-treated cells, for some unknown reason, did not detach from the plastic well by using trypLE (or trypsin). To evaluate the mitochondrial membrane potential by flow cytometry, the cells were therefore detached by using trypLE as described in 2.1, re-suspended the cells in complete cell culture medium (1 × 106/ml) and treated with rotenone (0.5 µM, 2 h) in an eppendorph tube. After exposure, the cells were washed once in PBS and stained with JC-1 and propidium iodide (5 µg/ml, Thermo Fisher) according to the protocol in serum free L-15 cell culture medium for 20 min at 19 °C. A minimum of 20 000 cells were then analysed by flow cytometry (Accuri C6, BD, Franklin Lakes, NJ, USA). The ratio of red fluorescence (Ex 488 nm/Em 564–606 nm) and green fluorescence (Ex 488 nm/Em 515–545 nm) were used to determine healthy versus depolarized mitochondria. Necrotic cells (PI positive cells; Ex 488 nm/Em 670 nm LP) were excluded from the analysis by gating.

ROS production was detected by using the oxidation-sensitive fluorescent probe, CM-H2DFDA (Molecular Probe, Invitrogen). The cells were first pre-incubated with CM-H2DFDA (2 µM) in PBS for 30 min, followed by exposure to rotenone in complete cell culture medium for 2 h. The medium was then replaced with HBSS and fluorescence (Ex 485 nm/Em 520 nm) analysed by Spectramax i3x plate reader. To ensure a representative readout of the fluorescent adherent cells, 37 different points were read in each well using the well scan function of the plate reader. Equal cell number in each well were verified by staining the nuclei with DRAQ5 (nuclear staining, 1:500; Thermo Fisher) for 30 min at room temperature, and cell number were counted by the spectramax i3x plate reader equipped with a microscopic module (MiniMax300Imaging Cytometer, Molecular Devices). For flow cytometric analyses, the cells were treated (CM-H2DFDA pre-incubation and rotenone exposure) in eppendorph tubes for reasons as described in 2.5. When using the efflux-pump inhibitor probenicid (1 mM), it was used during the CM-H2DFDA staining and rotenone exposure.

The relative concentrations of intracellular glutathione were determined by using using monobromobimane (mBBr), which binds to the SH group of the reduced form of glutathione (GSH), thereby forming a fluorescent adduct (Cotgreave and Moldeus 1986). After exposure, the cells were incubated with 40 µM mBrB (Sigma-Aldrich) in diluted in PBS with 2% FBS for 20 min at room temperature, and analysed by Spectramax i3x plate reader. To ensure a representative readout of the fluorescent adherent cells, 37 different points were read in each well by using the well scan function of the plate reader. For flow cytometry, the cells were trypsinated, resuspended in PBS and incubated with mBrB (20 µM) for 15 min. At least 10 000 cells were analysed by flow cytometry (Ex 405 nm/Em 515–545 nm) using NovoCyte Flow cytometer (Aglient, Santa Clara, CA, USA).

Catalase activity was measured by using Catalase Colorimetic Activity Kit (EIACATC Invitrogen, Thermo Fisher) as described by the manufacturer. Briefly, cells were seeded in 6 cm dishes as described above. The next day the cells were exposed for rotenone for 2 h, washed 2× with cold PBS and scraped in 250 µl cold Assay Buffer. The cell lysate was then frozen down (− 80 °C) until the next day. The cell lysate was then homogenizated by using a QIAshredder (Qiagen, Hilden, Germany), centrifuged (10,000×g, 15 min) and the supernatant collected. The assay was then further performed as described in the kit. Protein concentration of the catalase lysates were measured by using DC Protein Assay (Bio-Rad, Hercules, CA, USA).

The data analyses were performed using GraphPad Prism version 9.0.1(151). Statistical significance (p < 0.05) was assessed using ANOVA, followed by Holm–Sidak post-test. Standard error of the mean (SEM) is used as the mean of several independent experiments with three or more replicates are shown.