1. A Novel Role for c-Myc in G Protein–Coupled Receptor Kinase 4 (GRK4) Transcriptional Regulation in Human Kidney Proximal Tubule CellsNovelty and Significance
by John J. Gildea, Hanh T. Tran, Robert E. Van Sciver, Dora Bigler Wang, Julia M. Carlson, and Robin A. Felder
Hypertension
Volume 61(5):
April 17, 2013
Copyright © American Heart Association, Inc. All rights reserved.

2. Chromatin immunoprecipitation of c-Myc binding to the G protein–coupled receptor kinase 4 (GRK4) promoter.
Chromatin immunoprecipitation of c-Myc binding to the G protein–coupled receptor kinase 4 (GRK4) promoter. Cells were treated with phorbol 12-myristate 13-acetate (PMA; 100 nmol/L, 3 hours) or DMSO vehicle control (VEH). Degree of c-Myc binding was measured using qPCR of the GRK4 promoter. Copy number was greater for PMA-treated cells ( copies±100.25) than for VEH-treated cells ( copies±170.29), indicating a higher degree of c-Myc binding in response to PMA (>3 fold increase over VEH, n=3, P<0.05).
John J. Gildea et al. Hypertension. 2013;61:
Copyright © American Heart Association, Inc. All rights reserved.

3. A, The expression of phospho-c-Myc was measured by Western blot and graphed as the ratio of cellular actin after treatment with phorbol 12-myristate 13-acetate (PMA; 100 nmol/L, 24 hours) or DMSO vehicle (VEH).
A, The expression of phospho-c-Myc was measured by Western blot and graphed as the ratio of cellular actin after treatment with phorbol 12-myristate 13-acetate (PMA; 100 nmol/L, 24 hours) or DMSO vehicle (VEH). Phospho c-Myc expression was increased in response to PMA (127.78%±17.74, n=6, P<0.001). B, Western blot analysis showing G coupled–protein receptor kinase 4 (GRK4) expression was increased (40.51%±5.71, n=12, P<0.001) with PMA (100 nmol/L, 24 hours) and was blocked by the c-Myc inhibitor G5 (30 μmol/L, 24 hours). (+) control lane indicates lysates of human embryonic kidney (HEK) cells that have been transfected with a constitutively expressed GRK4. We used this lysate to confirm that the Western blot bands we analyzed were GRK4.
John J. Gildea et al. Hypertension. 2013;61:
Copyright © American Heart Association, Inc. All rights reserved.

4. ELISA for angiotensin (Ang) II
ELISA for angiotensin (Ang) II. Cells were treated with 3-Amino-4-thio-butyl sulfonate (EC-33; 500 nmol/L) or DMSO vehicle (VEH) for 3 hours.
ELISA for angiotensin (Ang) II. Cells were treated with 3-Amino-4-thio-butyl sulfonate (EC-33; 500 nmol/L) or DMSO vehicle (VEH) for 3 hours. The level of Ang II increased after three hours in VEH-treated cells compared with serum-free media only (SFM; 0.95 pg/mL±0.036 (n=6, #P<0.001), and in EC-33 treated cells vs SFM (1.40 pg/mL±0.050, n=6, #P<0.001). Angiotensin II also increased in EC-33–treated cells compared with VEH-treated cells (0.45pg/mL±0.050; n=6; *P<0.001)‏
John J. Gildea et al. Hypertension. 2013;61:
Copyright © American Heart Association, Inc. All rights reserved.

5. A, Phospho-c-Myc expression was increased in response to 3-Amino-4-thio-butyl sulfonate (EC-33; 500 nmol/L) by 75.00%±6.43 (n=6; P<0.001) and angiotensin (Ang) II (10 nmol/L, angiotensin receptor agonist) by 86.86%±15.21 (n=6; P<0.001).
A, Phospho-c-Myc expression was increased in response to 3-Amino-4-thio-butyl sulfonate (EC-33; 500 nmol/L) by 75.00%±6.43 (n=6; P<0.001) and angiotensin (Ang) II (10 nmol/L, angiotensin receptor agonist) by 86.86%±15.21 (n=6; P<0.001). The combination of Ang II and EC-33 caused phospho-c-Myc to increase by 79.54%±12.02 (n=6; P<0.001). There was no synergistic activity between the combination of EC-33 and Ang II, indicating that either agonist maximally stimulated phospho c-Myc levels. B, G coupled–protein receptor kinase 4 (GRK4) expression in response to EC-33, Ang II, and G5. The expression of GRK4 was increased by EC-33 (33.85%±6.16; n=6; P<0.01), Ang II (43.63%±10.11; n=6; P<0.001), and a combination of Ang II and EC-33 (34.12%±4.44; n=6; P<0.01). GRK4 expression was not increased with G5 alone or in combination with Ang II or all 3 agonists, indicating that G5 blocked the GRK4 agonist effects of EC-33 and Ang II.
John J. Gildea et al. Hypertension. 2013;61:
Copyright © American Heart Association, Inc. All rights reserved.

6. A, Phospho-c-Myc expression was decreased (21. 70%±4. 10; n=6; P<0
A, Phospho-c-Myc expression was decreased (21.70%±4.10; n=6; P<0.05) in response to losartan (LOS, 10 μmol/L; AT1R receptor antagonist) for 24 hours.
A, Phospho-c-Myc expression was decreased (21.70%±4.10; n=6; P<0.05) in response to losartan (LOS, 10 μmol/L; AT1R receptor antagonist) for 24 hours. B, G coupled–protein receptor kinase 4 (GRK4) expression decreased (18.49%±3.83; n=15; P<0.01) in response to Losartan (LOS, 10 μmol/L, 24 hours).
John J. Gildea et al. Hypertension. 2013;61:
Copyright © American Heart Association, Inc. All rights reserved.

7. The lack of response to fenoldopam (FEN)-stimulated cAMP accumulation in uncoupled RPTC (uRPTC) can be reverted to normal by incubation with the AT1R antagonist, losartan (LOS), or the c-Myc inhibitor G5.
The lack of response to fenoldopam (FEN)-stimulated cAMP accumulation in uncoupled RPTC (uRPTC) can be reverted to normal by incubation with the AT1R antagonist, losartan (LOS), or the c-Myc inhibitor G5. Intracellular cAMP accumulation was measured in real-time using a cAMP FRET biosensor, ICUE3. FEN stimulation (FEN, 1 μmol/L, 15 minutes) increased cAMP in normally coupled RPTC (nRPTC; *P<0.001 vs VEH; n=14) but not in uRPTC. Pretreatment of uRPTC with LOS (10 μmol/L, 3 hours) completely reverted the FEN-stimulated cAMP accumulation of uRPTC to normal (#P<0.001 vs uRPTC FEN; n=28). Similarly, pretreatment of uRPTC with the c-Myc inhibitor G5 (30 μmol/L, 3 hours) also completely reverted the FEN-stimulated cAMP accumulation of uRPTC to normal (#P<0.001 vs uRPTC FEN; n=47). Neither LOS nor G5 prestimulation significantly changed the response of nRPTC to FEN. Prestimulation of nRPTC with angiotensin II (Ang II, 10 nmol/L, 3 hours) caused the uncoupling of nRPTC (&P<0.001 vs VEH FEN, n=16). Similarly, prestimulation of nRPTC with phorbol 12-myristate 13-acetate (PMA; 100 nmol/L, 3 hours) also caused the uncoupling of nRPTC (&P<0.001 vs VEH FEN, n=14).
John J. Gildea et al. Hypertension. 2013;61:
Copyright © American Heart Association, Inc. All rights reserved.