Zen software version 3

Seedlings were imaged using a flatbed CanonScan 9950F scanner (Canon Inc., Öta, Japan) while they were growing on a plastic Petri dish. Histological detection of GUS activity and plant preparation for microscopy were performed according to Ditengou et al. [56 (link)]. For light microscopy, samples were observed using a Zeiss Axiovert 200M MOT device (Carl Zeiss MicroImaging, Jena, Germany) to capture high-magnification images. Low-magnification views were obtained using a Zeiss Stemi SV11 Apo stereomicroscope (Carl Zeiss MicroImaging) and observed under differential interference contrast optics. Plants expressing fluorescent proteins were stained with 5 µg/mL FM4-64, a lipophilic probe that binds to cell plasma membranes. The stained plants were then analysed using Nikon’s AZ-C1 Macro Laser Confocal Microscope and the Zeiss LSM 980 laser scanning microscope. To simultaneously monitor DAPI, YFP, and mCherry fluorescence, we employed multitracking in-frame mode, and the emission was separated using the META spectral analyser online unmixing feature. Images were extracted and analysed using Zen software version 3.4 (Carl Zeiss MicroImaging, Jena, Germany) and Imaris 9.8.0 (Oxford Instruments, Abingdon, UK).

Cells grown on cover slips were treated with OH-ME as indicated. DMSO served as solvent control, while N-OH-PhIP was included as positive control. Briefly, cells were fixed in ice-cold methanol at −20 °C for γH2AX staining or 4% paraformaldehyde (PFA) at room temperature for cytochrome c staining followed by blocking in PBS containing 5% (w/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X-100 for 1 h. Sample preparation for immunofluorescence staining of activated Bax included a fixation with 4% PFA followed by permeabilization with 0.2% CHAPS in PBS and a blocking step with PBS containing 5% (w/v) BSA. Immunofluorescence staining was essentially conducted as described [52 (link)] using the primary and secondary Alexa Fluor 488-conjugated antibodies listed in supporting Table S3. DNA was counterstained with TO-PRO-3 or DAPI (Life Technologies, Darmstadt, Germany) for 15 min. Finally, cells were mounted using VectaShield® (Vector Labs, Burlingame, CA) and analyzed by confocal microscopy using a Zeiss Axio Observer 7 microscope equipped with a 63x oil objective (Plan-Apochromat 63x/1.40 DIC M27) and a LSM900 confocal laser scanner (Zeiss, Oberkochen, Germany). Images were acquired by ZEN software version 3.4 (Zeiss) and processed using ImageJ software (NIH, MD).