Methods
In vitro studies
Female New Zealand White rabbits (1.8-2 kg) were purchased from Charles River (Oakwood, MI/Canada). RPTC were isolated using the iron oxide perfusion method and grown in 35-mm tissue culture dishes under improved culture conditions similar to what is observed in vivo (Nowak & Schnellmann, 1995, 1996). The culture medium was a 1:1 mixture of Dulbecco’s modified Eagles medium /F-12 (without glucose, phenol red or sodium pyruvate) supplemented with 15mM HEPES buffer, 2.5mM L-glutamine, 1µM pyroxidine HCl, 15mM sodium bicarbonate and 6 mM lactate. Hydrocortisone (50nM), selenium (5ng/ml), human transferrin (5µg/ml), bovine insulin (10nM) and L-ascorbic acid-2-phosphate (50µM) were added to fresh culture medium. Cells grown in the presence of glucose or mannitol were supplemented with 17mM D-glucose or 17mM D-mannitol (osmotic control). Confluent RPTC were used for all experiments.
GTP pulldown assay
GTP agarose beads (30µl) purchased from Abcam (Cambridge, MA) were washed three times with immunoprecipitation (IP) buffer (25mM Tris, 150mM NaCl) and incubated with 200µg protein overnight at 4oC with rotation. The following day, the bead-protein mixture was washed three times with IP buffer (25mM Tris Base, 150mM NaCl). Beads were boiled at 95oC with laemmli sample buffer for 5 min and the samples were centrifuged to harvest the pulldown proteins. GTP-bound proteins and sample input proteins were resolved on 4-15% SDS-PAGE gels, transferred onto nitrocellulose membranes and blotted for either Drp1, RhoA or Mfn1.
Immunoprecipitation
Dynabeads Protein G Immunoprecipitation kit (ThermoFisher, Waltham, MA) was used to immunoprecipitate RhoA. Proteins (200µg) were incubated with Pierce Protein A/G agarose beads (ThermoFisher) for 2 hr and centrifuged at 14,000g for 10 min at 4oC. Dynabeads were incubated with RhoA antibody (1:100) for 4 hr at room temperature. Supernatants from precleared lysates were added to the Dynabeads-RhoA antibody complex and incubated overnight at 4oC with rotation. RhoA was immunoprecipitated and eluted (denaturing elution) based on the manufacturer’s instructions. The resulting supernatant was loaded onto 4-15% SDS-PAGE gels with a 5% input control, transferred onto nitrocellulose membranes and blotted for p114RhoGEF. Membranes were incubated and visualized as described below.
Immunoblot analysis
Protein was extracted from RPTC cultures using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4). Protease inhibitor cocktail (1:100), 1mM sodium fluoride and 1mM sodium orthovanadate (Sigma-Aldrich, St. Louis, MO) were added fresh before each extraction. Equal protein quantities (10µg) were loaded onto 4-15% SDS-PAGE gels, resolved by gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with either 5% nonfat milk or 5% BSA in TBST and incubated overnight with primary antibody at 4°C with agitation. Primary antibodies used in these studies include Drp1 (1:1000) (#5991), RhoA (1:1000) (#2117) used for IP, pAkt (#9271), Akt (#9272) and GAPDH (1:1000) (#5174) were all purchased from Cell Signaling Technologies (Danvers, MA). Mfn1 (1:1000) (ab#2211661), p114RhoGEF (ab#96520) and RhoA (1:1000) (ab#187027) were purchased from Abcam. Membranes were incubated with the appropriate horseradish peroxidase conjugated secondary antibody before visualization using enhanced chemiluminescence (Thermo Scientific, Waltham, MA) and the GE ImageQuant LAS4000 (GE Life Sciences, Marlborough, MA). Optical density was determined using Bio-Rad Image Lab 6.0.
Analysis of oxygen consumption
Cultured RPTC were passaged onto 96-well XF96 extracellular flux analyzer plates (Agilent Technologies, Santa Clara, CA) at a cell density of 1.6x104 cells per well and grown in media containing 0mM glucose, 17mM mannitol or 17mM glucose for 96 hr. Cells were treated with pharmacological inhibitors at 72 hr after plating, for a period of 24 hr. Basal OCR was measured three times using the Seahorse Bioscience XF96 Analyzer before injection of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (2µM) (Sigma Aldrich) to measure OCR as previously described (Beeson et al., 2010). OCR was reported as picomoles per minute, and the results were normalized as a percentage of vehicle control (DMSO).
Data and statistical analysis
All data are shown as mean±SEM. Two-way analysis of variance followed by Tukey’s post hoc test was performed for comparisons of multiple groups. P<0.05 was considered statistically significant. All statistical tests were performed using the GraphPad Prism software (GraphPad Software, San Diego, CA). The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. Technical replicates were used to ensure the reliability of single values.