Supplementary Materials and Methods
Antibodies and reagents
CIB1 chicken polyclonal antibody was produced as previously described (1). Antibodies against
tubulin (TU-02), GAPDH (FL-335), PAK1 (N-20), Rb (IF8), ERK (K-23), cyclin E (M-20), and
cyclin D1 (DCS-6) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Caspase-7, caspase-9, lamin A/C, PARP, BAD, p-CDK1Tyr15, p-RbSer780, cyclin B1, p-GSK3Ser9,
pBADSer136, p-ERK, p-AKT308, p-AKT473, p-CDK2Thr160, p-H2AXSer139, p-CHK1S345, pan-AKT, MCL1, PUMA, BCL-2, HDAC1 antibodies and staurosporine were all obtained from Cell Signaling
Technology (Danvers, MA, USA). Mouse monoclonal anti-GAPDH and p-PAK1S199/204 were
purchased from Millipore. zVAD-FMK, JNK inhibitor SP00125 and AKTi-VIII were obtained from
EMD/Merck Gibbstown, NJ, USA). Deprenyl-R-(–)-hydrochloride (deprenyl) and iodoacetate
were purchased from Sigma (St. Louis, MO, USA).
Cell lines
The human SK-N-SH neuroblastoma and MDA-MB-468 breast cancer cell lines (UNC
Lineberger Tissue Culture Facility, UNC, Chapel Hill, NC, USA) were cultured in alpha Modified
Eagle’s
Medium
(MEM,
Gibco/Invitrogen,
Carlsbad,
CA,
USA)
and
DMEM/F12
(Gibco/Invitrogen), respectively, and 10% fetal bovine serum (FBS, Gemini Bio-Products, West
Sacramento, CA, USA). HEK293T cells were cultured in DMEM supplemented with 1% nonessential amino acids (Gibco/Invitrogen) and 10% FBS. All cells were maintained at 37°C in a
humidified atmosphere of 5% CO2.
Construction of lentiviral plasmids
Construction of control and CIB1 FG12 shRNA lentiviral plasmids was performed as previously
described (2). For this study, we also created a CIB1 shRNA-insensitive FG12 overexpression
plasmid (CIB1-sm) by introducing five silent, third base changes in codons 54 through 58
located within the 20 nucleotide CIB1 shRNA target.
The selected base substitutions
conformed to codon bias. Thus, two complementary primers containing the 20 nucleotide target
segment plus, respectively, 16 and 19 nucleotides 5' and 3' of this sequence, were used in a
standard QuikChange II (Agilent Technologies, Santa Clara, CA, USA) annealing and
polymerization protocol, with the exception that template DNA was used at five times the
recommended concentration.
FG12 lentiviral plasmids containing GAPDH derivatives were also constructed from parent
plasmids kindly provided by Akira Sawa (Johns Hopkins University School of Medicine). Briefly,
sequences of rat wild-type and mutant GAPDH-C150S were amplified by PCR from their
respective parent plasmids and cloned into a pBluescript-CMV shuttle vector (Stratagene); the
resulting CMV-GAPDH units were re-amplified and cloned directly into FG12 as previously
described (2). The resulting FG12-WT-GAPDH clone was also subjected to site-directed
mutagenesis (QuikChange II) to create an FG12-GAPD- K160R mutant derivative.
Lentiviral particle production and transduction of target cells
HEK293T cells were co-transfected with the HIV-1 lentiviral packaging constructs pRSVREV,
pMDLg/pRRE, pHCMVG, and FG12 expression vectors (3). The virus-containing supernatants
were harvested 24 h and 48 h after transfection and contaminating HEK293T cells were
removed by filtration through a 0.45 M filter. Target cells were infected with viral supernatants
containing 6 g/mL polybrene (Sigma) for 5 h and replaced with fresh growth media. For rescue
experiments and HA-GAPDH overexpression, target cells were transduced with CIB1-sm, HA-
GAPDH-WT, -C150 or -K160 shRNA lentivirus 24h prior to transduction with CIB1 shRNA.
Transduction efficiency was greater than 90% for all conditions as determined by GFP
fluorescence. Cells were harvested 72 h post transduction for all experiments.
Bromodeoxyuridine
(BrdU)
ELISA
cell
proliferation
assay
Proliferation of control and CIB1-depleted cells was determined using the BrdU ELISA cell
proliferation assay according to manufacturer’s instructions (Roche, Indianapolis, IN, USA) as
described previously (2).
Cell death quantification and DNA fragmentation assay
Adherent and floating cell populations were collected and cell death was quantified using trypan
blue dye exclusion and expressed as the mean percentage of dead cells (i.e., trypan blue
positive) from both floating and adherent cell populations. Statistical analysis was performed
using GraphPad Prism software.
Nucleosome formation was analyzed using the Cell Death
Detection ELISAplus kit according to manufacturer’s instructions (Roche). Briefly, SK-N-SH cells
were plated in 96-well plates (1X104 cells per well), cultured for 16-20 h, and adherent cells
were analyzed for DNA fragmentation/oligonucleosome formation using antibodies that detect
DNA-histone complexes.
Immunofluorescence
For analysis of GAPDH and AIF localization, cells were fixed with cold methanol, washed with
PBS and blocked with 3% normal goat serum. Cells were incubated with a monoclonal antiGAPDH or a rabbit polyclonal anti-AIF antibody and counterstained with DAPI and an Alexa488
conjugated secondary antibody. Fluorescent images were captured using ImagePro software
(Media Cybernetics, Bethesda, MD, USA) using a PlanApo 60X objective on an inverted Nikon
TE300 microscope.
Subcellular fractionation, cell lysis, and Western blotting
Subcellular fractionation was performed as described (4) with minor modifications. Briefly, cells
were washed twice in PBS and resuspended in ice-cold hypotonic lysis buffer (25 mM HEPES,
pH 7.5, 10 mM KCl, 1 mM EDTA, 0.2 mM pervanadate, 50 mM sodium fluoride, 10 mM βglycerophosphate, 10% v/v glycerol, supplemented with Protease Inhibitor cocktail Set III,
EMD/Merck) and incubated 10 min. on ice. Cells were lysed by addition of an equal volume of
hypotonic lysis buffer containing 0.2% v/v NP-40 followed by 15 min. incubation on ice.
o
Cytoplasmic fractions were collected after centrifugation at 3000 x g for 2 min. at 4 C and stored
o
at -80 C until further analysis. The nuclear pellet was washed once with hypotonic lysis buffer
with 0.1% NP-40 followed by resuspension in ice-cold hypertonic lysis buffer (25 mM HEPES,
pH 7.5, 420 mM NaCl, 10% w/v sucrose, 10% v/v glycerol, 10 mM KCl, 1 mM EDTA, 0.2 mM
pervanadate, 50 mM sodium fluoride, 10 mM β-glycerophosphate, and supplemented with
o
protease inhibitors) and incubated 30 min. at 4 C with constant rotation. Nuclear proteins were
o
isolated by centrifugation at 14,000 x g for 10 min. at 4 C and supernatants (nuclear fraction)
o
were stored at -80 C until further analysis.
SK-N-SH and MDA-468 total cell lysates were
prepared using CHAPS lysis buffer (50mM Hepes, 0.15 M NaCl, 5% v/v glycerol, 10 mM
CHAPS, 50 mM sodium flouride, 10 mM β-glycerophosphate, 0.1 mM sodium pervanadate, and
Protease Inhibitor Cocktail Set III). Lysates were not subjected to centrifugation prior to addition
of SDS-PAGE sample buffer to ensure no loss of protein for Western blot analysis. Lysates
were separated by SDS-PAGE, transferred to PVDF membranes, incubated with the indicated
primary antibodies overnight at 4° C and visualized using ECL or ECL plus (GE Healthcare,
Piscataway, NJ, USA).
JC-10 staining for mitochondrial membrane potential
Mitochondrial membrane potential was assayed using the Mito-ID Membrane Potential Kit
according to the manufacturer’s instructions (Enzo Life Sciences, Farmingdale, NY). Briefly,
cells were transduced with control or CIB1 lentivirus for 48 h as described above and seeded
onto black glass bottomed 96-well microtiter plates at 3 x 104 cells/well and cultured an
additional 24 h. Complete media was replaced with 100 l fresh media or media containing 4
M of the mitochondrial depolarizing agent, carbonyl cyanide 3-chlorophenylhydrazone (CCCP)
as positive control and cells incubated 30-45 min at 37°C. An equal volume of JC-10 dye
loading solution was added and incubated an additional 30 min at 37°C. Plates were read at
excitation/emission wavelengths 490/525 and 540/590 nm using a microplate reader
(Spectramax M5, Molecular Devices, Sunnyvale, CA).
Data is represented as relative
fluorescence units at 590 nM.
GAPDH activity assay
GAPDH activity was quantified using a GAPDH activity assay kit according to the
manufacturer’s instructions (Biomedical Research Service Center, Buffalo, NY).
This
colorimetric assay is based on the GAPDH-dependent reduction of iodonitrotetrazolium to
formazan via an NADH-coupled reaction.
Briefly, control and CIB1-depleted cells were
harvested and lysed in 1x lysis buffer and diluted to equivalent protein concentrations of 0.9
mg/ml with additional 1x lysis buffer. Cell lysates (15 l/well) were added to a 96-well microtiter
plate in duplicate and the enzymatic reaction initiated by the addition of 50 l GAPDH assay
solution to each well.
1x lysis buffer was used as background control.
The formation of
formazan was monitored at 492 nm for 1 h at 37°C in a microplate reader (SpectraMax M5,
Molecular Devices. The reaction was terminated with 50 l of 3% acetic acid and data is
represented as GAPDH activity/mg protein of optical density readings at 492 nm (absorbance
maximum, A492).
Statistical analysis
Statistical analyses represent the means of at least 3 independent experiments. Error bars
represent the standard error of the mean (SEM). P values were calculated using Student’s t test
and results reaching a p value of 0.05 (*p>0.5) were considered significant. All calculations
were performed using Prism software (GraphPad, v. 5.0, San Diego, CA, USA).
Supplementary References
1.
Leisner TM, Liu M, Jaffer ZM, Chernoff J, Parise LV. Essential role of CIB1 in regulating
PAK1 activation and cell migration. JCell Biol. 2005;170(3):465-76.
2.
Zayed MA, Yuan W, Leisner TM, Chalothorn D, McFadden AW, Schaller MD, et al. CIB1
regulates endothelial cells and ischemia-induced pathological and adaptive angiogenesis.
CircRes. 2007;101(11):1185-93.
3.
Qin XF, An DS, Chen IS, Baltimore D. Inhibiting HIV-1 infection in human T cells by
lentiviral-mediated delivery of small interfering RNA against CCR5. ProcNatlAcadSciUSA.
2003;100(1):183-8.
4.
Milosevic J, Bulau P, Mortz E, Eickelberg O. Subcellular fractionation of TGF-beta1stimulated lung epithelial cells: a novel proteomic approach for identifying signaling
intermediates. Proteomics. 2009;9(5):1230-40.