Nis elements v 3

Isolated cells (2,000–3,000 in 5–10 µl of IMDM) were pipetted onto poly-L-lysine coated slides (P0425-72EA; Sigma-Aldrich), settled down for 15 min at RT, fixed with 4% paraformaldehyde for 10 min at RT, then washed three times with PBS, and permeabilized/blocked for 1 h at RT with 0.1% Tween-20 in 10% FBS (Corning) in PBS, which was then used as antibody incubation buffer for all the subsequent steps. Cells were incubated overnight at 4°C with a mouse monoclonal anti-KDEL (ab12223; Abcam) or a rabbit anti-mouse β-catenin (9582S; Cell Signaling) primary antibody, washed three times with PBS, and incubated for 1 h at RT with a goat anti-mouse IgG A488 (A11029; Invitrogen) or a donkey anti-rabbit-A555 (A31572; Invitrogen) secondary antibody. Cells were then washed three times with PBS, stained with 1 μg/ml DAPI (32670; Sigma-Aldrich) for 10 min at RT, washed three times with PBS, and finally slides were mounted with VectaShield (H-1000; Vector Laboratories). Cells were imaged on a Nikon Ti Eclipse inverted confocal microscope with 60× objective using Nikon NIS Elements (v3) for data collection, and images were processed using Fiji (https://fiji.sc). Cells were imaged on an Olympus epifluorescence microscope with 60× objective for manual scoring of nuclear β-catenin staining. At least 100 cells per condition were randomly captured for quantification.

Autophagy flux was measured using the tandem fluorescence LC3-GFP adenovirus. CMs were measured in Tyrode solution (140 mM NaCl, 5.4 mM KCl, 1.8 mM Ca²⁺,1 mM MgCl₂, 10 mM HEPES, 5.6 mM glucose, pH = 7.3 using NaOH). Cells were infected for 48 h with 1 MOI of adenovirus containing TF-LC3-GFP. Z-stack images were taken in 1-μm steps through the cell. Two channels per stack were collected. GFP was excited using a 460–500 nm filter and emission was collected from 500 to 560 nm. RFP was excited using a 540–580 nm filter and emission was collected at 600–660 nm. Focused images were created in NIS Elements V3 (Nikon Instruments). Pixels ratio = 1 represent the autolysosomal region. Total areas of ratio >1 were calculated with respect to total surface area to measure autophagy flux.

Endothelial cell migration was studied as previously described (Alvarez-García et al., 2013a (link)). Briefly, HUVEC control or melatonin treated for a week cells were cultured into 6-well plates (Falcon) in VCBM supplemented with 2% FBS and were allowed to reach full confluence. With a pipette tip a line of cells was scraped away. Then, cells were washed with PBS and irradiated. In the plates, four randomly selected views along the scraped line were photographed using an ORCA R2 camera (Hamamatsu Photonics, Massy Cedex, France) attached to a microscope set Nikon Ti (Werfen Group, Barcelona, Spain) at 10 × magnification. Photomicrographs were taken every 10 min during the course of the experiment, which was terminated as soon as the wound was completely filled in vehicle treated controls (after 10 h). Initial and final wound sizes were measured using the Nis Elements v.3.8 software (Nikon, Tokyo, Japan) and the difference between the two was used to determine migration distance using the following formula: initial wound size minus final wound size divided by two.

HUVEC control or melatonin 1 mM, treated for a week, cells were cultured into 6-well plates (Falcon) in VCBM enriched with 2% FBS and were allowed to reach full confluence. Based in previous works^{23 (link)}, a line of cells was scraped away in each plate using a pipette tip. Cells were washed with PBS and docetaxel 10 nM or vinorelbine 10 nM or its diluent (ethanol at a final concentration lower than 0.001%) was added to each plate. After that, four randomly selected views along the scraped line in each plate were photographed using an ORCA R2 camera (Hamamatsu Photonics, Massy Cedex, France) attached to a microscope set Nikon Ti (Werfen Group, Barcelona, Spain) at 10x magnification. Photomicrographs were taken every 10 minutes until the wound was completely filled in controls (after 10 hours). Initial and final wound sizes were determined using the Nis Elements v.3.8 software (Nikon, Tokyo, Japan) and the difference between the two was used to determine migration distance using the following formula: initial wound size minus final wound size divided by two.

MDA-MB-231 and MCF-7 cells were seeded into 6-well plates (Life Sciences, Tewksbury, MA, USA) and cultured for 24 h in DMEM supplemented with 10% FBS (csFBS) to reach full confluence. A line of cells was scraped away in each plate using a pipette tip. Media were replaced by fresh ones containing melatonin (1 nM) and/or doxorubicin (1 μM) or vehicle (ethanol at a final concentration lower than 0.0001%). At least four randomly selected views along the scraped line in each plate were photographed using an ORCA R2 camera (Hamamatsu Photonics, Massy Cedex, France) attached to a microscope set Nikon Ti (Werfen Group, Barcelona, Spain) at 10× magnification. Microphotographs were taken every ten minutes during the course of the experiment, which was terminated after 24 h. Initial and final wound sizes were determined using the Nis Elements v.3.8 software (Nikon, Tokyo, Japan) and the difference between the two was used to determine migration distance as follows: initial wound size minus final wound size divided by two. Each assay was performed at least three times.