Nis elements ar v4

Cells were seeded on adherent coverslips (Sarstedt) and sterilized scaffolds for 3 and 6 days. After the PBS rinsing, cells were fixed with 4% (w/v) paraformaldehyde, permeated using 0.2% (v/v) Triton X-100, blocked using 3% (w/v) BSA, and dyed the actin cytoskeleton by rhodamine-phalloidin (1:250) and the nucleus by DAPI (1:1000). Fluorescence was examined through a Nikon A1R confocal laser scanning microscope (CLSM) (Nikon, Tokyo, Japan) and all images were taken by Nikon NIS-Elements AR v4.10 software (Nikon). Image J (National Institutes of Health, Bethesda, MD, USA) software was used to determine nuclear and cytoplasmic elongation. As described elsewhere [40 (link)], five cells from each image were randomly chosen to measure the length and width of the nucleus and cytoplasm.

NCI-H1975 cells were seeded on adherent coverslips (Sarstedt) or PCL scaffolds for 3 and 6 days. Samples were fixed using 4% paraformaldehyde solution (weight/volume), permeated by 0.2% Triton ™ X-100 (volume/volume), blocked by 3% BSA solution (weight/volume), and dyed using rhodamine-phalloidin (1:250) and DAPI (1:1000). Fluorescence was observed under an A1R confocal laser scanning microscope (CLSM; Nikon, Tokyo, Japan). Images were taken through Nikon NIS-Elements AR v4.10 software (Nikon). Nuclear and cytoplasmic circularity was determined using MATLAB software (MathWorks). At least three replicates of each type of PCL scaffold were made at 3 and 6 days. For each replicate, three representative images were taken. Subsequently, a software was implemented to count the number of cells, calculate the sample size knowing the size of the population, and randomly choose the cells from which the perimeter and area were calculated. Then, the circularity was calculated with the following formula: $$Circularity=\frac{4\ast \pi \ast area}{{\mathit{perimeter}}^2}$$
The equation was designed to give values around 0 for an elongated nucleus or cytoplasm, whereas a perfect circle will give a circularity value of 1.

All images were quantified using Nikon NIS- Elements AR v4.40. To determine Rif1 and SUUR signal intensities at sites of gene amplification, Regions Of Interest (ROIs) were identified based on the EdU intensity. SUUR or Rif1 mean signal intensity was then determined within each ROI. Ten randomly selected regions outside of the nucleus were selected and the mean signal intensity for these regions were averaged to determine the background signal for each image. The average background signal was subtracted from the signal at amplified regions to normalize each image for varying amounts of background. To quantify the SUUR signal intensity at heterochromatin, SUUR ROIs were manually defined due to the their non-uniform shape. The sum intensity of the fluorescent signal within these regions were extracted. The sum signal intensity was then normalized to ROI area to account for the difference in shape of each ROI. To quantify PLA signals, ROIs were generated based on DAPI signal to mark all nuclei. PLA foci were then identified for each image and the number of foci in each DAPI ROI was determined.

Cells were harvested, diluted into PBS (81 mM Na₂HPO₄ + 24.6 mM NaH₂PO₄ + 100 mM NaCl) and 1 μl of each culture was placed on a pad of 1% agarose made up in 0.5X TAE buffer. Images were collected using a Nikon Eclipse Ti inverted microscope equipped with an Orca Flash 4.0 digital camera (Hamamatsu), with a Nikon TIRF 1.45 NA Plan Neoflur 100 oil immersion objective. NIS-Elements AR (v 4.40, Nikon) software was used to collect and analyze images, which were then imported into Photoshop to configure the images for publication. Fluorescence intensities were determined using the automated General Analysis tool of NIS-Elements.