Confocal dishes

Cells were seeded in confocal dishes (SPL Life Sciences, Korea), and were treated with different conditions as described in results. The cells were fixed with Fixative (Science Cell, US) for 15 min. After permeabilization with Triton X-100, the cells were blocked in 1% BSA (Sigma-Aldrich, US) for 30 min, and incubated with primary antibody (1/50 dilution) overnight at 4 °C, and then secondary antibodies (1/400 dilution) for 1 h. Images were captured with Confocal microscopy (LEICA TCS SP8, Leica Microsystems, Germany) and quantitative analysis was performed with ImageJ software.

Cells were seeded into confocal dishes (SPL, Korea) at a density of 20 000 cells per ml. After 12 h, cells were treated with 0.1% DMSO (as control) or compound PBQC (1, 5 and 10 μM) for 12 h, cell apoptosis rate was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay.³³ Briefly, cells were fixed with cold 4% paraformaldehyde for 25 min and incubated with 0.2% Triton X-100 for 5 min at room temperature after washed with 1× PBS three times, discarded the supernatant and then washed the plates with 1× PBS twice. Added 100 μL equilibration buffer into each hole for 10 min, then, poured off the supernatant and incubated cells with 50 μL rTdT incubation medium that was away from light for 60 min at 37 °C. After incubating for 1 h, we discarded the incubation medium and added 50 μL 2× SSC into the plates for 15 min to terminate the reaction. After washing cells with PBS three times, stained cells with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and observed and photographed by a confocal microscopy (Carl Zeiss, Germany), the excitation wavelength is 488 nm.

HDFs were seeded in confocal dishes (SPL Life Sciences, Pocheon-si, Korea) and cells were processed. Next, 0.5 μM Lysosensor™ Green DND-189 (pH = 5.2) (Invitrogen, Carlsbad, CA, USA) treated cells for 30 min, and 1× PBS washed three times. Confocal laser scanning microscope 700 (Carl Zeiss, Jena, Germany) was used to detect the fluorescence intensity. The excitation wavelength of Lysosensor™ Green DND-189 is 443 nm.

Cells were cultured in confocal dishes with diameters of 35 mm (SPL, Seoul, Korea) and fixed with 4% PFA in PBS, permeabilized for 5 min with 0.1% (v/v) of Triton X-100, and washed at each step three times with PBS for 5 min. The cells were blocked with 5% normal goat serum (NGS, Vector Lab, Burlingame, CA, USA) in PBS for 30 min. Anti-AR antibody was diluted in 5% (v/v) NGS in PBS for 4 h at RT. After washing with PBS three times for 5 min, the cells were incubated with Alexa Fluor 555 secondary antibodies (Invitrogen) for 1 h at room temperature and counterstained with DAPI (Invitrogen). Cells were visualized with a Andor SRRF system (Oxford instruments, Abingdon, UK). For analyzing ER-mitochondrial contacts, cells were treated with C3A and DHT for 24 h. After washing the cells with PBS three times, cells were incubated with Mito-Tracker green (200 nM) and ER-Tracker (200 nM) in supplement-free medium for 20 min at 37 °C, and nuclei were stained with DAPI.

ES2 and OV90 cells (3 × 10⁴ cells per 300 μL) were prepared on confocal dishes (SPL Life Science, Gyeonggi-do, Korea), and then 50 µM eupatilin was treated for 48 h at 37 °C in a CO₂ incubator. After treatment, the cells were air-dried and fixed with 4% paraformaldehyde for 1 h at 25 °C. The cells were briefly rinsed with PBS and permeabilized by 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Then, the cells were stained by tetramethylrhodamine (TMR) red, for 1 h at 37 °C in the dark. Cells were then washed with PBS and overlaid with DAPI. Fluorescence was assessed using an LSM710 (Carl Zeiss, Oberkochen, Germany) confocal microscope fitted with a digital microscope AxioCam camera with Zen2009 software (Carl Zeiss, Oberkochen, Germany).

For bright-field imaging, a SteREO Discovery V20 stereomicroscope (Zeiss) or a BX50 microscope (Olympus) was used. For confocal imaging, embryos were mounted in 1% low melting temperature agarose (Lonza) on confocal dishes (SPL Life Sciences) and imaged under an LSM 510 Pascal, or an LSM 700 confocal laser scanning microscope (Zeiss). Images were acquired by Zen Black (version 8.1, Zeiss) and assembled using Adobe Photoshop (version CS6). An SP8 intravital multiphoton microscope (Leica) at Korea Basic Science Institute was used to image beating motile cilia in zebrafish embryos, and images were captured using LAS X (Leica)