Uncovering the protective role of lipid droplet accumulation against acid-induced oxidative stress and cell death in osteosarcoma

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Highlights • Lipid droplet formation mitigates cellular stress • Targeting key proteins involved in lipid droplet formation leads to a significant decline in cell viability and increased ROS production • Lactate is a key metabolite in lipid droplet formation • Lipid droplet biogenesis is fuelled by tumor-associated stromal cells Files show ROS quantification, measured on plate reader, by means of DCF fluorescence. The file named ROS time points, shows raw data and graph of ROS measurment in 143B cultured in normal 7.4 medium (time point 0) or in acid 6.8 medium at different time points (2, 7 and 24h). ROS were identified by the 2′,7′-dichlorofluorescin (DCFH) method. In summary, cells were cultured in complete medium at 7.4 or 6.8 pH. At the indicated time points, cells were washed and then incubated with 10 μM 5- and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Monza, Italy) for 5 minutes at 37 °C. The fluorescent signal of DCF resulting from the transformation of 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) by intracellular ROS production was measured at 485–535 nm using a microplate reader (Tecan Infinite F200pro, Mannedorf, Switerzland). The results were expressed as the mean of the relative fluorescence units (RFU). The file named ROS with siRNA shows ROS quantification after silencing of DGAT1 or PLIN2, two key components of lipid droplet formation. Silenced cells were transferred into 500 uL/well of pen/strep-free medium in 96-well plates for ROS quantification (5,000 cells/well). 24 hours after electroporation, cells were transferred in complete medium at 7.4 or 6.8 pH for the indicated times.

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