Incucyte nuclight rapid red reagent

For staining of lipid droplets, we used HCS LipidTOX following the manufacturer’s instructions (Invitrogen). For live imaging of cells as described for fatty acids up-take we treated cells at day 5 of differentiation with BODIPY (10 μM) C₁₂ (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid) (Invitrogen). In addition, to measure cell growth cells were stained with Incucyte NucLight Rapid Red Reagent for nuclear labeling following the manufacturer’s instructions (Sartorius). Those experiments were monitored using Incucyte live cell analysis system. For immunostaining 7 days post differentiation, cells were plated on coverslips for 24 hr and following fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, rinsed with PBS, and permeabilized with 0.2% Triton X−100 in PBS for 5 min at room temperature. Blocking was done with normal goat serum for 1 hr. Following primary antibody phospho-HSL (Ser660) 1:100 (Cell signaling) was added for overnight incubation. All samples were then incubated in secondary antibody 1: 1000, Alexa Fluor 488 conjugate (Abcam), and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen).

AC16 human cardiomyocytes (MilliporeSigma, SCC109) were seeded onto a 96‐well plate at 8000 cells per well in 100 μL of culturing media. Next day, cells were treated with IncuCyte NucLight Rapid Red Reagent (1:500; Sartorius, Ann Arbor, Michigan; 4717) and Caspase‐3/7 Green (1:1000; Sartorius, 4440) in culture medium alone or plus OptiMEM, or plus AMSC‐derived conditioned media, or plus Brachyury transfected AMSC‐derived conditioned media. Concomitantly, 20 μM of LY83583 (Cayman chemical, Ann Arbor, Michigan; 70230) was added. Nucleus positive for Caspase‐3/7 were counted every 2 hours up to 24 hours (IncuCyte S3 Live‐Cell Analysis System, Sartorius). Superoxide dismutase (SOD) activity and total antioxidant capacity of conditioned media were measured using respective kits (Abcam, Cambridge, United Kingdom; ab65354 and ab65329). The concentration/ratio of conditioned medium ranged between 1.69 and 6.05 μg/μL, depending on the protein assay applied. Throughout protocols, the concentration was consistent among groups with equivalent volume of conditioned media loaded per well.

ETV2-ECs and ETV2-SPI1-ECs were cultured until day 12 of differentiation on Matrigel-coated wells in a 24-well plate. BALB/c LSECs were isolated and plated as well on collagen at 200,000 cells per well in a 24-well plate in 0% FBS-DMEM. After 1 h of LSEC plating, ETV2-ECs, ETV2-SPI1-ECs, and LSECs were supplemented with 10 µg/ml FSA-FITC (prepared as described in ref. ^{47 (link)}) and 1:1000 IncuCyte® NucLight Rapid Red Reagent (cat. 4717, Sartorius). Cells were imaged hourly over a period of 24 h using the IncuCyte Cell analysis system (Essen Bioscience). Quantification was performed with the IncuCyte Cell analysis software. This experiment was repeated twice. Statistical differences were assessed by mixed ANOVA.

NCI-H358 cells were seeded into T75 flasks (1 × 10⁶ cells per flask) and the following day the media was replaced with media supplemented with the indicated concentration of inhibitor. Resistant cell populations were generated using a constant IC₉₀ high concentration of inhibitors (2.63 µM ARS1323, 830 nM ARS1620, 87 nM selumetinib, 2.34 µM AZD4785). Media changes were carried out every 2–3 days for small molecule inhibitors and every 4–5 days for AZD4785 until the cells reached 80% confluency. The cells were then trypsinised and re-seeded at the original density and aliquots of the remaining cells were prepared for liquid nitrogen storage. The development of emerging resistance was monitored by shifts in the cell viability dose responses using IncuCyte® Nuclight Rapid Red Reagent (Sartorius). To examine the effects of drug withdrawal from resistant cells, the day after seeding cells were washed with media only and then treated with media containing 0.1% DMSO. Cells were split as required or media changed every 2–3 days until confluent.

Cell proliferation was determined using the IncuCyte^® Live-Cell Analysis system (Essen Bioscience). Cell proliferation was either determined as measurement of cell density (confluence) or by quantification of red nuclei using the IncuCyte^® NucLight Rapid Red Reagent (Essen Bioscience). Staining was performed according to manufacturer’s instructions. Alternatively, viable cell number was determined by trypan blue staining and automatic counting using a Countess™ Cell Counting Chamber Slide (Thermo Fisher).

For automated live cell proliferation experiments, the cells were seeded onto 96-well plate: 30 000 VCaP cells and 5 000 22Rv1 cells per well (±ENZ 3w). On the day 0 (d0) of monitoring, the nuclei were stained with IncuCyte NucLight Rapid Red reagent (Essen BioSciences, #4717). The cells were monitored every 8 h in total of 6 days using IncuCyte S3 Live-Cell Imaging System (Essen BioSciences). IncuCyte S3 2021C software (Essen BioSciences) was used to count the number of cells. Results were normalized to d0 and are shown as relative percentage in cell count, representing the mean ± SD of 8 images. For MTS assay, the cells were seeded onto 96-well plates: 30 000 VCaP cells and 10 000 22Rv1 cells per well in charcoal-stripped medium (±ENZ). For RNA interference, the cells were transfected with siNON or siFOXA1 as described above. Wells containing only medium were used to subtract background. The start of the treatments was designated as d0. The cell proliferation was assessed at d0, d1, d2, d3 and d4 using colorimetric CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) kit (Promega, #G3580) according to manufacturer's protocol. The absorbance was measured at 492 nm wavelength using Multiskan EX plate reader (Thermo Scientific). Results were normalized to d0 and are shown as fold change. P-values were calculated using two-way ANOVA with Bonferroni post hoc test.