Pathway 855

Cells were seeded at 10,000 and 15,000 cells per well in 96 well clear bottom imaging plates (353219, Falcon). After 24h, the medium was refreshed with plain medium or medium containing 10 μM BENSpm. After another 24h, cells were washed with PBS and fixed for 5 minutes using 10% neutral buffered formalin (HT5011, Sigma). After removing the formalin cells were washed with PBS followed by a blocking/permeabilization step using PBS 1% Bovine Serum Albumin, 0.1% Triton X-100 (staining buffer). Cells were stained by incubation with various anti-SMOX antibodies at 0.1–3 ug/mL for one hour. Primary antibodies were removed by washing the wells three times with 150 μL PBS, 0.1% Triton X-100 (wash buffer). Secondary staining was achieved by incubating the cells with 1 ug/mL AlexaFluor 488 labeled anti-rabbit IgG (A11034, Invitrogen) and 10 ng/mL DAPI in staining buffer. After one hour the wells were washed three times with 150 μL wash buffer followed by the addition of 100 μL PBS per well. The wells were imaged using a High Content imager (Pathway 855, Becton Dickinson) with a 20x objective taking 4 images per well per channel. Alexa Fluor 488 and DAPI images were stitched and overlaid using ImageJ software (version 1.53g10).

For immunofluorescence microscopy, the automated microscope Pathway 855 (Becton Dickinson, Franklin Lakes, NJ, United States) was used to read fluorescence intensity in 96-well plates. For confocal microscopy, LSM 510 laser scanning microscope (Carl Zeiss, Germany) was used.
The cells were fixed in 3.7% paraformaldehyde for 20 min, followed by permeabilization with 0.5% triton-X in PBS for 15 min and blocking for 15 min using blocking solution (3% BSA in PBS). The primary antibody to phospho-H2AX (05-636, Millipore)/phospho-Rad17 (6981, Cell Signaling Technology)/CtIP (61142, Active Motif), diluted in blocking solution, was added for 1 h, followed by incubation with a secondary antibody (Alexa-Fluor 546/488) and Hoechst 33342 (Invitrogen) diluted in blocking solution for 45 min.
Images were captured and analyzed using the BD Pathway software, wherein the region of interest (ROI), in this case the cell nuclei, were defined by Hoechst stain, and the average intensity of the antibody-coupled fluorescence within each ROI was determined.

C2C12 myoblasts were seeded in 384-wells black/clear imaging plates at a density of 4,000 cells/well, 24 hours prior to ibipinabant treatment. A 1,000× concentrated serial √10-dilution in DMSO was made for ibipinabant and the positive control etoposide. Immediately before use, ibipinabant and etoposide stock solutions were diluted 100× in phosphate buffered saline (PBS) and 10× in the culture medium, resulting in a final DMSO concentration of 0.1% (v/v). Four replicates of each compound were tested up to maximum solubility (100 μM). After 24 h and 48 h exposure, nuclei were stained for cell viability analysis using Hoechst 33342. After 20 minutes of staining at 37 °C, fluorescence was imaged on a BD Pathway 855 high-throughput microscope (Becton Dickinson (BD) Bioscience, Breda, The Netherlands). Followed by the analysis of the number of nuclei using Cellprofiler33 (link).

The cells were cultured on a black 96-well plate. The confluent fibroblast cells were treated as described in Section 3.1.2. After 24 h, the culture media were removed and the cells were fixed with a 3.7% formaldehyde solution at room temperature for 10 min. Then, the plate was washed once with 100 µL/well of PBS. Later, the permeabilization with a 0.1% Triton X-100 solution and a 10 min step was performed. After the permeabilization, the plate was washed twice with PBS and 3% FBS was used as a blocking agent at room temperature for 30 min. After the FBS removal, 50 µL of the primary antibody (1:50), diluted in 3% FBS, was added and the plate was incubated for 1 h at room temperature. After the incubation, the primary antibody plate was washed three times with PBS. Then, 50 µL per well of the secondary antibody (dilution 1:1000) was added for the next 1 h. During this step, the plate was covered from the light. When the secondary antibody solution was removed, the plate was washed 3 times with PBS and the wells were filled with 100 µL of PBS containing 2 µg/mL of Hoechst 33342 for the nuclei staining. The plate was visualized using a confocal laser scanning microscope, BD Pathway 855 (Bioimager, Becton Dickinson, Franklin Lakes, NJ, USA), supported with AttoVision^TM 1.6 software.

As the PrestoBlue assay measures mitochondrial activity, we confirmed that the effects of metformin, phenformin and chloroquine on cell viability were caused by an absolute decrease in cell number by fixing the cells in 4% paraformaldehyde for 15 min, followed by nuclear staining using Hoechst 33342 (Fischer Scientific). The plates were imaged using a BD Pathway 855 imager (Becton Dickinson), after which the images were processed using an Image-Pro Analyser 7.0 algorithm. Hoechst area was used as a read out to quantify the amount of cells in each well.

Cells seeded in 96-well plates were fixed with PFA 4% for 10 minutes at RT. After washing with PBS, they were permeabilized with PBS-Triton 1% for 5 minutes at RT, washed with PBS-BSA 2%, and incubated with BODIPY 493/503 (dilution: 1/40) (Invitrogen Molecular Probes, Carlsbad, CA, USA) for 2 hours in the dark at RT. They were finally incubated with Phalloidin-Alexa 555 (dilution: 1/50) (Invitrogen Molecular Probes, Carlsbad, CA, USA) and Hoechst FluoroPure grade (dilution: 1/1000) (Invitrogen Molecular Probes, Carlsbad, CA, USA) for 30 minutes in the dark at RT and analyzed using the BD Pathway 855, with the AttoVision software and BD-IDE software (lens: 20x) (Becton Dickinson, Franklin Lakes, NJ, USA).

The cells were cultured on a black 96-well plate. When the cells reached 80% confluency, an AmO was added and UVA irradiation was performed (as described in Section 3.2.2). After 24 h, the culture media were removed and the cells were fixed with a 3.7% formaldehyde solution at room temperature for 10 min. Then, the plate was washed once with 100 µL/well of PBS. Later, the permeabilization with a 0.1% Triton X-100 solution and a 10 min step was performed. After the permeabilization the plate was washed twice with PBS and 3% FBS was used as a blocking agent at room temperature for 30 min. After the FBS removal, 50 µL of the primary antibody (1:50), diluted in 3% FBS, was added and the plate was incubated for 1 h at room temperature. After the incubation, the primary antibody plate was washed three times with PBS. Then, 50 µL per well of the secondary antibody (dilution 1:1000) was added for the next 1 h. During this step, the plate was covered from the light. When the secondary antibody solution was removed, the plate was washed 3 times with PBS and the wells were filled with 100 µL of PBS containing 2 µg/mL of Hoechst 33342 for the nuclei staining. The plate was visualized using a confocal laser scanning microscope BD Pathway 855 (Bioimager, Becton Dickinson, Franklin Lakes, NJ, USA) supported with AttoVision^TM 1.6 software.