Fig. 3
ACSL3 inhibition induces cell death in ccRCC. A XTT cell viability assay of ccRCC cells cultured in media supplemented with standard FBS or CSFBS and treated with increasing concentrations of triacsin C. n = 3, error bars depict standard deviation from the mean. B XTT cell viability assay of ccRCC cells cultured in atmospheric oxygen (21% O2, Nx) or hypoxia (0.5% O2) and treated with increasing concentrations of triacsin C. n = 3, error bars depict standard deviation from the mean. C Cell titer blue assay of cell viability in ccRCC cells transfected with siACSL3 or non-targeting control siRNA. n = 3, error bars depict standard deviation from the mean. D Representative western blots of ACSL3, cleaved caspase 3 and β-actin from ccRCC cells transfected with siACSL3 or non-targeting control siRNA. E Densitometry quantifications of ACSL3 western blots using actin as a loading control. n = 3, error bars depict standard deviation from the mean. Comparisons are from an unpaired two-tailed Student’s t test comparing control cells with cells transfected with siRNA against ACSL3. *p < 0.05. F FACS assay of cleaved caspase 3 in ccRCC cells transfected with siRNA against either ACSL3 or non-targeting control. n = 3 with 2 technical replicates representing 10,000 cells per technical replicate. Comparisons are from an unpaired two-tailed Student’s t test comparing the mean fluorescence intensity of cleaved caspase 3 positive cells in the two groups. **p < 0.01. G Cell titer blue viability assay of ccRCC cells pre-treated with the caspase inhibitor, zVAD.FMK followed by treatment with increasing concentrations of triacsin C. H Orthotopic ccRCC xenograft model of tumor weight in mice injected with ccRCC cells. Mice were injected with ccRCC cells treated with shRNA against either ccRCC or negative control. n = 25. 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 from an unpaired two-tailed Student’s t test comparing the control cells with the shACSL3 cells grouped together. **p < 0.01