Fig. 1

Exogenous lipids are the major source of lipid droplet formation in ccRCC. A Oil Red O stained RCC4 cells cultured in media supplemented with or without glucose, glutamine (2 mM), pyruvate (1 mM) and with 10% complete fetal bovine serum (FBS) or charcoal stripped FBS (CSFBS). Images displayed at the same magnification. Imaging was performed with n = 2 biological replicates. B Quantification of 1A. The box plots summarize the distribution of the amount of Oil Red O staining in each cell, relativized to the average control cell. The whiskers were produced using the Tukey method. Individual points represent values that fall outside the range used in the Tukey method. The edges of the boxes represent the 25th and 75th percentile, while the line in the center of the box represents the mean. Comparisons are multiple comparisons from a two-way ANOVA using Tukey’s multiple comparisons test, comparing the column effect between each group on the X-axis. P(CSFBS vs FBS) = 0.0135, p(FBS vs no pyruvate) = 0.3887, p(FBS vs no glutamine) = 0.4771. The row effects (comparisons between each level of glucose) were not significant. p > 0.05. C Oil Red O stained control and VHL-transduced RCC4 cells cultured in media supplemented with 10% CSFBS ± 100 μM oleic acid for 24 h prior to staining. Images displayed at the same magnification. Imaging was performed with n = 2 biological replicates. D Quantification of 1C. The box plots summarize the distribution of the percent area of Oil Red O staining in each cell, relativized to the average control cell. The whiskers were produced using the Tukey method. Individual points represent values that fall outside the range used in the Tukey method. The edges of the boxes represent the 25th and 75th percentile, while the line in the center of the box represents the median. Comparisons are multiple comparisons from a two-way ANOVA using Tukey’s multiple comparisons test, comparing all groups. P(RCC4 + oleic vs RCC4 + VHL + oleic) = < 0.0001. P(RCC4 + oleic vs RCC4 + CSFBS) = < 0.0001. E LC-MS measurement of triglyceride abundance in control and VHL-transduced RCC4 cells cultured in media supplemented with 10% CSFBS ± 100 μM oleic acid for 24 h prior to staining. n = 3, error bars depict standard deviation from the mean. F LC-MS ¹³C stable isotope tracing using universally labelled glucose, glutamine, or palmitate to measure fractional labeling of triglycerides containing 3 palmitate acyl groups. The number of labelled carbons measured is depicted on the x-axis. n = 3, error bars depict standard deviation from the mean