To pro 3

U937-PR9 cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) medium (Sigma Aldrich R7388) added with 1% penicillin/streptomycin (Sigma-Aldrich P4333) and 10% fetal bovine serum (Sigma-Aldrich F9665) and maintained at 37 °C and 5% CO₂. U937-PR9 were seeded on poly-l-lysine (Sigma-Aldrich P8920) coated glass coverslips before experiments. Cells were fixed with 4% (w/v) paraformaldehyde (PFA) at room temperature for 10 min. Next, cells were washed with 3% BSA and then permeabilized with 0.5% (v/v) Triton X-100 for 20 min.
For TO-PRO-3 staining, samples were extensively washed with PBS and stained with TO-PRO-3 (T3605-Thermo fisher scientific) at a dilution 1:1000 in BB, for 12 min at room temperature. For immunofluorescence experiments, cells were blocked with 3% BSA in PBS and incubated in a wet chamber with a RNA polymerase II CTD repeat (phospho S2) rabbit primary antibody (ab5095 Abcam) diluted in Incubation Buffer, overnight at 4 °C. Cells were then extensively washed with Washing Buffer 3 × 15 min and incubated with a Goat α-rabbit Atto 647N secondary antibody (40839 Sigma-Aldrich) diluted in IB for 1 h at room temperature, followed by the same washing procedure with WB. After washing with PBS, coverslips were mounted on glass slides with ProLong Diamond Antifade Mountant (Invitrogen P36961).

Aggregates were fixed in cold (-20°C) methanol-acetone 1 : 1 (v/v) for 20 minutes and washed with PBS. They were then incubated, by shaking with MF20 anti-myosin antibody, (from hybridoma culture medium, diluted 1 : 5) at 4°C for 20 h. Washed aggregates were incubated with fluorescein-labelled secondary antibodies for 2 h at room temperature. Nuclei were stained with TO-PRO-3 (0.2 mg/ml, Molecular Probes, Molecular Probes Europe, Rijnsburgerweg, The Netherlands). Samples were observed by confocal microscopy with a Leica TCS SP2 (Leica, Wetzlar, Germany).

Embryos were fixed and treated as described in [26 (link)]. The primary antibody was polyclonal anti-Phospho-Smad 1/5 (Cell Signalling; 1:10), the secondary was Alexa Fluor 488 Goat Anti-Rabbit IgG (Invitrogen; 1:500), and nuclear staining was done with TO-PRO3 (Molecular Probes; 10 μM). Fluorescently labeled embryos were mounted in DAKO or 3:1 glycerol/PBS. Confocal images were collected using the Confocal Microscope C2+ (Nikon) and processed using NIS-Elements Microscope Imaging Software (Nikon) and FIJI image analysis software [12 (link)].

Normal and tumor cells were seeded at a density of 2×10⁵ on glass coverslips and left to adhere overnight at 37°C in 5% CO2. In the first part of experiment, the cells were exposed to 250μM of 6-AN for 24h, or incubated in medium alone, as previously described. In the second part of experiment, the cells were exposed to 250μM of 6-AN for 24h and then were treated with 100 μM of ascorbic acid 2-phosphate (AA2P) for 24h. After several washes to remove AA2P the cells were incubated for 72h. To study mitochondrial superoxide generation, cells were stained with 5 μM MitoSOX red for 10 min at 37°C and washed three times before imaging. Cells were fixed using ice-cold 4% paraformaldehyde for 10 min at room temperature. Then, nuclei were revealed by counterstaining with TO-PRO-3 (Molecular Probes). Images were taken with a Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope using an ×63 objective lens.

Slice cultures were left attached on the Millicell membrane and stained as free-floating sections in 6-well plates. Cultures were first fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) (PAA, Austria) for 2 h at 4°C. After washing with PBS, slices were permeabilized by 0.1% TritonX-100/PBS for 90 min at RT. Slices were then blocked with 5% bovine serum albumin (BSA) for 2 h and afterwards incubated with primary antibody diluted in PBS for 3 days at 4°C. After washing three times with PBS, slices were incubated with secondary antibody for 2 days at 4°C. Finally, slices were incubated with the nuclear counterstain and dead cell indicator TO-PRO3 (Molecular Probes, US) for 15 min and washed three time with PBS before getting mounted with Permafluor mounting solution (Beckman Coulter, Paris, France. The following primary antibodies were used: pan-Tau antibody K9JA (Dako, Hamburg, Germany, Nr. A0024 (1:1000)), 12E8 (1:1000) for phosphorylated S262/S356 Tau (gift from Dr. P. Seubert, Elan Pharma, South San Francisco, CA, US) and Synaptophysin (1:1000; Sigma). All fluorescent (goat anti-rabbit/mouse cyanine 2 and 3)-labeled secondary antibodies were from Dianova (Hamburg, Germany) (1:1000).

To evaluate DNA damage, si-RNA-transfected cells were pretreated with 100 μM AHR-inh for 1 h, then exposed to CSC or DMSO for 6 or 8 h. DNA fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) as reported previously^{56 (link)} using the in situ cell death detection kit as per the manufacturer (Roche Diagnostics, Indianapolis, IN, USA). In other experiments, spermatocytes were pretreated with 100 μM AHR-inh for 1 h, treated with CSC for 17 h, and stained with FITC mouse anti-cleaved Poly(ADP-ribose) polymerase (PARP) antibody (BD Biosciences, San Jose, CA, USA) and counter-stained with TO-PRO-3 (Molecular Probes) (1:1,000, 5 min) at room temperature. Cells were then analyzed by flow cytometry.