Co2 incubator

The cells (1 × 10⁵ cells/ml) from each group were cultured in each well of the 24-well plate and imaged every 30 min for H69AR cells or every minute for SHP77 cells in live up to desired time point. Images were taken with a 10X objective on an EVOS cell imaging microscope with a CO₂ incubator (Thermo Fisher Scientific). For inhibitor treatment, each inhibitor at the desired dose (see the resources table for details) was added at the beginning of imaging for 24 h. Analyses were all done using FIJI’s trackmate. Cell motility was analyzed by measuring the position of the cell centroid at every time point and plotted to show the trace of centroid movement. The distance that the cell centroid traversed for each time point was calculated to determine the level of the movement.

Primary neonatal rat CM (NRCM) cultures were performed as described previously^{72 (link)}. Briefly, 1- to 3-day-old Sprague–Dawley rats were killed by swift decapitation according to the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health). NRCMs were isolated from the hearts of these mice and seeded into 6-well culture plates at a density of 1 × 10⁶ cells/well in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% FCS and 1% penicillin/streptomycin. Following a 48-h incubation, the culture medium was replaced with DMEM containing 0.1% FCS and AraC (0.1 mm) (Sigma), after which the NRCMs were infected with Adβ-catenin (Adβ-cat), AdGFP, Adshβ-catenin (Adshβ-cat) and AdshRNA (Cyagen Biosciences, Santa Clara, CA) at a multiplicity of infection of 10. Transgene expression was detected in 95% to 100% of the cells. Next, the NRCMs were exposed to a 1% O₂ environment in a CO₂ incubator (Thermo Fisher Scientific) for 48 hours to induce a hypoxia cell model. Control cells were cultured under normoxic conditions.

The ability of the extracts to enhance or suppress RAW 264.7 cell proliferation was evaluated by XTT{2,3‐bis (2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide innersalt} assay kit (Welgene Inc). RAW 264.7 cells were seeded in 96‐well plates at a concentration of 1 × 10⁶ cell in 89% Dulbecco's modified Eagle's medium (DMEM from Thermo Fisher Scientific Solutions LLC) containing 10% fetal bovine serum (v/v) and cultured until 100% confluence. Each sample was diluted to 50, 100, and 200 μg/ml. XTT reagent (1 ml) and 20 μl PMS reagent (N‐ethylphenazonium methyl sulfate) were used to prepare a working solution. Equal volumes of the cultured cell supernatant and the working solution (100 μl) were put into the 96‐well plate and were incubated for 4 hr in a CO₂ incubator (Thermo Scientific). Absorbance was measured using a microplate reader (Tecan GENios FL Fluorescence Microplate Reader FI TRF, USA). The absorbance at 450 nm was subtracted from the absorbance at 690 nm. The ability of the extracts to promote or suppress the growth of the cells was interpreted as their cytotoxicity (Figure 3).