Fig. 4
Metabolism of exogenous lipids sensitizes ccRCC cells to ferroptosis inducers. A Cell titer blue viability assay of MDA-RCC-62 cells cultured in media supplemented with standard FBS or CSFBS and treated with 5 μM erastin. n = 3 biological replicates, error bars depict the standard deviation from the mean. Comparisons are multiple comparisons from a one-way ANOVA using Å Ãdák’s multiple comparisons test to measure the difference between erastin and untreated cells within each media group. ****p < 0.0001. B FACS assay of MDA-RCC-62 cells stained with POPRO membrane permeable dye to mark dying cells. Cells were cultured in media supplemented with either standard FBS or CSFBS and treated with 2.5 μM erastin. Bars represent the mean value and error bars represent the standard deviation. Results are from twelve replicates performed over two independent experiments. Comparisons are multiple comparisons from a one-way ANOVA using Å Ãdák’s multiple comparisons test to measure the differences between all groups. F = 14.54 with 8 degrees of freedom. P(FBS + erastin vs CSFBS + erastin) = 0.0046. C Cell titer blue viability assay of MDA-RCC-62 cells cultured with BSA, or BSA + 100 μM oleic acid, linoleic acid, or arachidonic acid and treated with the indicated concentration of erastin. D Cell titer blue viability assay of MDA-RCC-62 cells cultured in media supplemented with 10% CSFBS + BSA, or BSA + 20 μM arachidonic acid, or BSA + 20 μM arachidonic acid + 1 μM triacsin. n = 4 biological replicates, error bars depict the standard deviation from the mean. E Cell titer blue assay of MDA-RCC-62 cells treated with 1 μM triacsin C or vehicle control and increasing concentrations of erastin. n = 3 biological replicates, error bars depict the standard deviation from the mean. F Cell titer blue viability assay of MDA-RCC-62 cells transfected with siACSL3 or non-targeting control siRNA and treated with increasing concentrations of erastin. n = 3 biological replicates, error bars depict the standard deviation from the mean. G FACS analysis of BODIPY 493/503 fluorescent intensity in ccRCC cells cultured with only BSA, added 100 μM oleic acid, or oleic acid and DGAT inhibitor cocktail (DGATis). Bars represent the mean value and error bars represent the standard deviation. Results are from 30 replicates performed over 4 independent experiments. Comparisons are multiple comparisons from a one-way ANOVA using Å Ãdák’s multiple comparisons test to measure the differences between all groups. F = 86.21 with 27 degrees of freedom. P(BSA vs oleic) = < 0.0001. P(oleic vs oleic + DGATis) = 0.0005. H Cell titer blue viability assay of MDA-RCC-62 cells cultured with either BSA or 100 μM oleic acid alone or supplemented with either erastin or erastin and DGATis. Results are presented as a violin plot, where the width of the shape shows the distribution of the data. The dashed line represents the median where the dotted lines represent the quartiles. Results are from 48 replicates performed over 2 independent experiments. Comparisons are multiple comparisons from a two-way ANOVA using Å Ãdák’s multiple comparisons test to measure the differences between oleic and BSA within each group (row effect). P(BSA + erastin vs oleic + erastin) = < 0.0001. P(BSA + erastin + DGATis vs oleic + erastin + DGATis) = < 0.0001