Incucyte flr live cell analysis system

For figures 1A and 1C, 350 cells/well were plated into 96-well plates and treated as indicated in the figures (n=60 wells / condition). Medium with fresh drugs was changed every 3–5 days. The confluency of the wells was determined weekly using the Incucyte FLR live cell analysis system (Essen Bioscience). For figure S1B, cells were plated and treated as above, and the wells were manually scored as positive when the confluence was above 50 % and assessed weekly (Tricker et al., 2015 (link)). For figure S1C, 78 000 PC-9 cells were plated into T25 flasks and treated the next day as indicated. Cell proliferation was monitored using Incucyte HD live cell analysis system (Essen Bioscience) by imaging 32 sectors in the T25 flask. For all other long-term growth assays (≥10 days), 1000 cells/well were plated into 96-well plates and treated as indicated in the figures (n=5–12 wells / condition). The confluency of the wells was determined daily using Incucyte HD. Endpoint cell viability assays were performed using Cell Titer Glo (Promega) according to manufacturer’s instructions.
To determine number of dormant cells after treatment, viable cells were manually counted from the Incucyte images. A total of 10–12 wells with 3 images per well was analyzed for each condition.

Cells were plated at 2,000–4,000 cells/well in 96-well plates in either standard culture media or serine/glycine-free media, as indicated. Starting the day of plating, cellular proliferation was monitored in an Incucyte FLR Live Cell Analysis System (Essen BioScience) in a humidified incubator containing 5% CO₂ at 37°C. For studies involving NCT-503, PHGDH Inactive, or GNE-618, cells were treated the day after plating with the indicated concentrations of compounds in the indicated media conditions. Experiments were performed at least 3 times.