Fig 1.
NR4A1 plays a role in RCC proliferation.
(A) Pathways activated by NR4A1 and targeted by NR4A1 antagonists. (B) Cells were transfected with two different oligonucleotides targeting NR4A1 [NR4A1(1) and NR4A1(2)] and after 72 hr, the number of cells were determined as outlined in the Materials and Methods. (C) Cells were treated with DIM-C-pPhOH and DIM-C-pPhCO2Me and cell numbers were determined after treatment for 24 hr. (D) Cells were transfected with NBRE3-luc and 40 ng FLAG-NR4A1, treated with DMSO, DIM-C-pPhOH (20 μM) and DIM-C-pPhCO2Me (15 μM), and luciferase activity was determined as described [17]. (E) Cells were transfected with siCtl (non-specific) or siNR4A1 and treated with DIM-C-pPhOH and DIM-C-pPhCO2Me and cell numbers were determined. siNR4A1 alone decreased cell proliferation as indicated in (B) and this value was set at 100% to determine the effects of C-DIMs in cells after loss of NR4A1. (F) Athymic nude mice bearing ACHN cells as xenografts were treated with 30 mg/kg/d DIM-C-pPhOH and tumor volumes were determined. Results (C—E) are means ± SE for at least 3 separate determinations and significantly (p < 0.05) decreased growth/volume is indicated (*). Significant (p < 0.05) attenuation of C-DIM-induced growth or luciferase activity or after transfection with siNR4A1 is also indicated (**).
Table 1.
Antibodies and sources.
Fig 2.
NR4A1 knockdown and C-DIM/NR4A1 antagonists induce apoptosis in RCC cells.
ACHN (A) or 786-O (B) cells were transfected with siNR4A1(1) and siNR4A1(2) and Annexin V staining was determined as outlined in the Materials and Methods. ACHN (C) and 786-O (D) cells were treated with 20 μM DIM-C-pPhOH or DIM-C-pPhCO2Me for 24 hr and Annexin V staining was determined. Results are means ± SE for 3 replicated determinations and significant (p<0.05) induction of Annexin V staining is indicated (*).
Fig 3.
C-DIM/NR4A1 antagonists target nuclear NR4A1.
ACHN (A) and 786-O (B) cells were treated with 20 μM DIM-C-pPhOH and DIM-C-pPhCO2Me. Cells were immunostained with NR4A1 antibodies or DAPI and images merged as outlined in the Materials and Methods.
Fig 4.
NR4A1 plays a role in expression of Sp-regulated growth promoting and survival genes.
(A) Cells were transfected with siNR4A1 and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. Cells were treated with DIM-C-pPhOH (B) or DIM-C-pPhCO2Me (C) and after 24 hr, whole cell lysates were analyzed by western blots. (D) Western blot analysis of tumor lysates from athymic nude mice bearing ACHN xenografts and treated with vehicle (control) or DIM-C-pPhOH (30 mg/kg/d) was also determined. Band intensities were quantitated relative to β-actin (means ± SE) and significantly decreased staining intensities are indicated (*; p<0.05).
Fig 5.
siNR4A1 and C-DIM/NR4A1 antagonists induce stress in RCC cells.
Cells were transfected with siNR4A1 (A) or treated with DIM-C-pPhOH (B) or DIM-C-pPhCO2Me (C) and whole cell lysates were analyzed by western blots. (D) After treatment of cells as described in (A)—(C), ROS was measured using the cell permeant CM-H2DCFDA probe as outlined in the Materials and Methods. Significant induction (p<0.05) of ROS is indicated (*). (E) Tumor lysates from athymic nude mice bearing ACHN cells as xenografts and treated with vehicle control or DIM-C-pPhCO2Me were analyzed by western blots and individual bands were quantitated (relative to β-actin). Significant (p<0.05) induction of ROS is indicated (*).
Fig 6.
siNR4A1 and NR4A1 antagonist inhibit mTOR in ACHN cells.
Cells were transfected with siNR4A1 (A) and treated with DIMI-C-pPhOH (B) and DIM-C-pPhCO2Me (C), and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods.
Fig 7.
siNR4A1 and NR4A1 antagonist inhibit mTOR in 786-O cells.
Cells were transfected with siNR4A1 (A) and treated with DIMI-C-pPhOH (B) and DIM-C-pPhCO2Me (C), and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. (D) Induction of sestrin 2 by western blots was determined in 786-O cells treated with DIM-C-pPhOH and DIM-C-pPhCO2Me or transfected with siNR4A1 in the presence or absence of 5 mM GSH.