FIGURE 1 from Sigma1 Regulates Lipid Droplet–mediated Redox Homeostasis Required for Prostate Cancer Proliferation

Abstract

<p>Sigma1 is required for DHT-induced AR-mediated LD accumulation in prostate cancer cells. <b>A,</b> Confocal micrograph showing LD accumulation in LNCaP cells cultured in CSS containing medium for 3 days and then treated for 1, 2, 3, and 6 days of DHT (1 nmol/L). HCS LipidTOX stained LDs (red). DAPI stained nuclei (blue). Quantification of LD number per cell and average area of LD particles/cell. Data represent mean values from at least three independent determinations, and error bars represent SEM. LD particle numbers and lipid area were quantified using ImageJ. Statistical analysis was performed using ANOVA and Bonferroni after test. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001<b>. B,</b> Confocal micrograph showing LD accumulation in LNCaP transduced with nonspecific control and Sigma1 shRNA. Two distinct Sigma1 shRNA clones were tested and produced comparable results. Cells were cultured in CSS containing medium for 3 days and then treated for 3 days with DHT (1 nmol/L). LD number per cell was determined as in A, above. <b>C,</b> Immunoblots of whole-cell protein extracts from LNCaP cells infected with Sigma1 shRNA #4 and #5 and treated, serum starved for 3 days, and treated with 3 days of DHT. <b>D,</b> SRS confirming lipid content of LDs in LNCaP cells following 3 days of 1 nmol/L DHT treatment in CSS medium, conditions described above. <b>E,</b> LD numbers per cell in panel of AR-driven (C4-2, C4-2B), ARV-driven (22Rv1), and AR-negative, independent (PC3, DU145) prostate cancer cell lines. Data represent mean values from at least three independent determinations, and error bars represent SEM. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.0001; ns = no significance. <b>F,</b> Confocal micrograph of LDs in PC3 cells (endogenous AR-negative prostate cancer cell line), transfected with empty vector (pcDNA) or recombinant AR plasmid, then treated with DHT (1 nmol/L, 3 days). Quantification of the average number of LDs per cell. Right, Quantification of the mean number of particles per cell ± SE. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01. <b>G,</b> Confocal micrograph showing that LDs accumulate only in AR-transduced PC3 cells. AR (green), LDs (red), DAPI stained nucleus (blue). <b>H,</b> Immunoblot further confirming transduction and expression of recombinant AR in PC3 cells. <b>I,</b> ARV7-induced LDs require Sigma1. LDs (red) in 22Rv1 cells transduced with nonspecific control shRNA or Sigma1 shRNA #5. Magnified inset (white boxes) shown below. LD stain (red), DAPI stain (blue). <b>J,</b> Immunoblot confirmation of Sigma1 shRNA KD in 22Rv1 cells. <b>K,</b> Control confirming that only ARV7-positive cells are also LD-positive. ARV7 immunostain (green), LD stain (red), DAPI stain (blue). <b>L,</b> LDs (red) in PC3 cells transduced with nonspecific control shRNA or Sigma1 shRNA #5 and subsequently transfected with ARV7. Magnified inset (white boxes) shown below. DAPI stain of nuclei (blue). Average number of LDs per cell calculated and analyzed as above. <b>M,</b> Immunoblot confirmation of Sigma1 shRNA KD and transfected ARV7 expression in PC3 cells.</p>