Mitotracker deep red 633

For confocal microscopy experiments, BT474 cells were plated on 35-mm Petri dishes (15,000 cells/cm²) and treated with appropriate drugs as indicated in the figure legends and/or in the text. Cells rinsed three times with 2 mL of HBSS buffer were incubated in 2 mL of HBSS buffer, supplemented with 2 µg/mL Hoechst 33342 (H3570, Invitrogen, Waltham, MA, USA), SYBR Green I at dilution of 1:200,000 (S7563, Invitrogen, USA), and 150 nM MitoTracker Deep Red 633 (M-22426, Molecular Probes, Eugenius, OR, USA) at 37 °C for 30 min in a CO₂-free thermostat. Following staining, the cells were rinsed 3 times with dye-free HBSS, and fluorescent images of cells were obtained using fluorescent scanning confocal microscope Leica TCS SP-5 DM6000 CS (Leica Microsystems, Wetzlar, Germany) at a sequential scanning mode using HCX PL APO lambda blue 63×, NA = 1.4 (Leica Microsystems, Wetzlar, Germany). Excitation and emission were set for Hoechst 33342 405 nm/460 nm, SYBR Green I 488 nm/540 nm, and MitoTracker Deep Red 633 633 nm/710 nm. Quantification of the number of mitochondrial nucleoids and mitochondrial size was performed using the fluorescence confocal images of BT474 cells labeled for mtDNA (stained with SYBR Green-1) and mitochondrial mass (stained with MitoTracker Deep Red). Images were analyzed using the Fiji software algorithm [52 (link)] and macros described in [12 (link)].

After fixation and permeabilization, NRCM (1×10⁴ cells/mL) or female heart tissue sections (in 5 μm thickness) were stained with anti-p38β primary antibody (sc-6187-R, Santa Cruz) at room temperature for 1 hr. Following triple wash in PBS, they were incubated with Fluorescein anti-rabbit IgG (V0729, Vector laboratories) for 30 min in dark, rinsed in PBS three times, then co-incubated with 1 mM MitoTracker deep red 633 (Molecular Probes) to stain mitochondria. Heart tissue slices were mounted with mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI), and observed under confocal microscope (Zeiss) for p38β (green fluorescence), mitochondria (red) and nucleus (blue).

In situ hybridization was performed as previously [28 (link)]. Mitochondria were labelled in live cells by incubation for 5–30 min in 0.1 µM TMRE (tetra-methylrhodamine ethyl ester; Invitrogen) or Mitotracker Deep Red 633 (Molecular Probes) in 0.2 µm Millipore-filtered seawater. Fluorescence emission spectra of Clytia tissues and life-cycle stages were obtained without coelenterazine pre-incubation using a Leica SP5 confocal microscope. Measurements were performed on live adult medusae or eggs mounted between slide and coverslip in seawater. These were excited with a 488 nm argon laser line and images acquired at successive 3 nm spaced, 5 nm wide bands between 475 and 625 nm using the xyλ scanning mode. Quantification was then performed on several ‘regions of interest’ defined to sample particular structures: manubrium, bell, tentacle bulb, gonad ectoderm and eggs. Data were acquired from multiple regions of two separate medusae with similar spectra obtained for each tissue. Additional fluorescence images were obtained using an Olympus epifluorescence microscope.

Following differentiation, IPEC-J2 were treated with DON (30–100 μM) for 24 h and subsequently incubated with 25 nM MitoTracker Deep Red 633 (Life Technologies, Carlsbad, CA, USA) for 30 min, fixed with 3.7% formaldehyde in PBS and counterstained with 150 nM 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA) in PBS. Images were captured using a laser scanning confocal microscope (Leica SP5 II, Wetzlar, Germany).

MitoTracker Deep Red 633 (Life Technologies, Carlsbad, CA, USA) was used to stain mitochondria by confocal microscopy as previously described [21 (link)]. Mitochondrial number was also monitored by flow cytometry. Cells were incubated with MitoTracker Deep Red 633 (200 nM) for 20 min at 37 °C and immediately analysed by a FACSCanto flow cytometer (Becton-Dickinson).

Mouse anti-CFAP52 polyclonal antibody (aa 421–620) was generated by Quan Biotech and was used at a 1:500 dilution for Western blotting. Rabbit anti-CFAP45 antibody (HPA043618, Atlas Antibodies) was used at a 1:500 dilution for Western blotting and a 1:400 dilution for immunofluorescence. Mouse anti-GAPDH antibody (AC002, ABclonal) was used at a 1:5000 dilution for Western blotting. Rabbit anti-Tubulin antibody (A6830, ABclonal) was used at a 1:5000 dilution for Western blotting. Mouse anti-GFP antibody (M20004, Abmart) was used at a 1:2000 dilution for Western blotting. Rabbit anti-MYC antibody (BE2011, EASYBIO) was used at a 1:2000 dilution for Western blotting. Rabbit anti-AKAP4 antibody (A14813, ABclonal) was used at a 1:100 dilution for immunofluorescence. Mouse anti-acetylated α tubulin antibody (T7451, Sigma-Aldrich) was used at a 1:200 dilution for immunofluorescence. The secondary antibodies were goat anti-rabbit FITC (1:200, ZF-0311, Zhong Shan Jin Qiao), goat anti-mouse FITC (1:200, ZF-0312, Zhong Shan Jin Qiao), and goat anti-rabbit TRITC (1:200, ZF0313, Zhong Shan Jin Qiao). MitoTracker Deep Red 633 (1:1500 dilution, M22426, Thermo Fisher Scientific) was used for immunofluorescence.