Fig. 2

ACSL3 is required for lipid droplet accumulation in ccRCC cells. A Acyl-CoA synthetase catalyzes the esterification of free fatty acids with coenzyme A, a step that is necessary for downstream anabolic and catabolic metabolism of fatty acids. B Images of Oil Red O stained ccRCC cells treated with the pan-ACSL competitive inhibitor, triacsin C. Images displayed at the same magnification. Imaging was performed with greater than n = 3 biological replicates. C Quantification of the ORO staining in MDA-RCC-62 cells (see Fig. 1B). 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. The comparison is an unpaired two-tailed t test with 165 degrees of freedom. P = < 0.0001. 170 cells were measured over the course of three independent replicate experiments. D qPCR measurement of mRNA expression of ACSL subspecies in MDA-RCC-62 cells transfected with either siRNA against the indicated ACSL subspecies or a control scrambled siRNA (siCTRL). ACSL6 was not found to be expressed in ccRCC cells and therefore was not included in this figure. Bar graph represents mean and error bars represent standard deviation. All knockdowns were performed 3 times per experiment in 2 independent experiments. E Images of Oil Red O stained ccRCC cells transfected with siRNA against ACSL1, 3, 4, 5, and 6 cultured with 100 μM oleic acid for 24 h. Images displayed at the same magnification. Imaging was performed with n = 2 biological replicates. F Images of Oil Red O stained ccRCC cells transfected with siACSL3 and cultured with 100 μM linoleic acid, arachidonic acid, or control media for 24 h prior to fixation and staining. Images displayed at the same magnification. Imaging was performed with n = 2 biological replicates. G Oil Red O stained ccRCC cells treated with either scrambled control siRNA or siRNA against ACSL3, treated with either charcoal stripped FBS or charcoal stripped FBS supplemented with 100 μM oleic acid. Images displayed at the same magnification. Imaging was performed with n = 2 biological replicates. H Quantification of 2G. 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. 287 cells were counted over 3 independent replicate experiments. Comparisons are multiple comparisons from a two-way ANOVA using Šídák’s multiple comparisons test to measure the differences between all groups. P(siCtrl + oleic vs siACSL3 + oleic) = < 0.0001. P(siCtrl + oleic vs siCtrl + CSFBS) = < 0.0001