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

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.

研究亮点
• 脂滴（Lipid droplet）形成可减轻细胞应激
• 靶向参与脂滴形成的关键蛋白，可显著降低细胞活力并增加活性氧（ROS）生成
• 乳酸是脂滴形成过程中的关键代谢物
• 肿瘤相关基质细胞可为脂滴生物发生（Lipid droplet biogenesis）提供能量支持

本数据集包含通过酶标仪检测的DCF荧光法定量活性氧的相关结果。其中名为"ROS时间点"的文件，展示了在正常pH 7.4培养基（时间点0）或酸性pH 6.8培养基中培养的143B细胞，在不同时间点（2、7和24小时）的活性氧检测原始数据与对应图表。活性氧通过2′,7′-二氯荧光素（DCFH）法进行检测。具体实验流程如下：将细胞于pH 7.4或pH 6.8的完全培养基中培养，至指定时间点后，洗涤细胞并于37℃下用10 μM的5-及6-羧基-2′,7′-二氯二氢荧光素二乙酸酯（CM-H2DCFDA; 赛默飞世尔科技，意大利蒙扎）孵育5分钟。细胞内活性氧可将2′,7′-二氯二氢荧光素二乙酸酯（DCFH-DA）转化为DCF，通过多功能酶标仪（Tecan Infinite F200pro，瑞士曼内多夫）在485–535 nm波长下检测其荧光信号，结果以相对荧光单位（RFU）的均值表示。

名为"siRNA干预ROS检测"的文件，展示了沉默脂滴形成的两个关键组分——DGAT1与PLIN2后的活性氧定量结果。将沉默后的细胞以每孔5000个的密度接种于无青霉素/链霉素的完全培养基中，铺于96孔板，每孔体积500 μL用于活性氧检测。电穿孔24小时后，将细胞转移至pH 7.4或pH 6.8的完全培养基中培养指定时长。