Bloom syndrome is a
condition that affects multiple systems in the body and is associated with
growth deficits, skin problems, compromised immune system, insulin resistance
and increased risk of developing diabetes and cancer (1).
A preliminary analysis
was carried out to determine whether proteins are differentially expressed in
patients with Bloom syndrome. SILAC mass spectrometry analysis of wildtype
fibroblasts and fibroblasts isolated from patients with Bloom syndrome showed
that several proteins are upregulated in affected individuals (Shastri and
Schmidt, unpublished observation). Out of the different proteins that showed a
statistically significant upregulation, the protein of interest in my study is
Factor A (TFAM) is a nuclear encoded mitochondrial DNA transcription factor (2).
Specific binding of TFAM to the light strand promoter and the heavy strand
promoter regulates transcription and replication of mitochondrial DNA (3). TFAM
also functions in the compaction, organization and stabilization of the mtDNA
through its high mobility group (HMG) box domain. By modulating multiple
functions, TFAM can regulate mitochondrial copy number. Studies have shown that
TFAM protein levels directly correlate to mitochondrial copy number (4) (5) (6).
From the initial screen
it is evident that TFAM is upregulated in fibroblasts isolated from patient
with Bloom syndrome. Previous studies have indicated that oxidative stress
biomarkers are upregulated in Bloom syndrome (7) (8) (9). The goal of my
project is to determine whether TFAM mediated increase in mitochondrial number
is the contributing factor to the oxidative stress phenotype that is observed
in patients with Bloom syndrome.
GMO 0637, GMO 8505, KHS
1452 and KHS 1453 fibroblast cell lines were cultured at 37°C and 5% CO2 in
Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 1%
For lysate preparation,
cells were washed with ice cold PBS. Cells were harvested in ice cold RIPA
lysis buffer using cell. The lysate was then vortexed continuously for 30 min
at 4 ºC
. The mixture was then centrifuged at 14000 rpm for 15 min and supernatant was
collected in fresh tube.
The protein lysates
were then normalized for equal amounts of proteins using Pierce BCA protein
assay kit. Samples were prepared using 2X Laemmli buffer containing 5%
?-mercaptoethanol. It was then boiled at 96 ºC for 5 min and loaded onto 10%
SDS-PAGE gels. The gels were run at constant voltage and the proteins were
transferred to 0.45µm PVDF membranes by wet transfer at 4 ºC for 90 min at
320mA. The membrane was blocked for an hour in 5% of Skimmed milk prepared in
TBST (Tris-Buffered Saline supplemented with 0.05% Tween 20) followed by
incubation with primary antibody (prepared in 5% BSA in TBST, Dilution 1:1000)
against protein of interest overnight at 4 ºC . Bound primary antibody was
recognized using horseradish peroxidise coupled secondary antibody which was
incubated at room temperature for 1hr. Following each step, the blots were washed
three times in TBST for 5 min each. Chemiluminescence was detected and the
images were acquired using ChemiDoc MP System (Bio-rad).
cells were plated on sterile coverslips placed in 6 well plate.
staining, cells were fixed using 4% Paraformaldehyde at 37 ºC for 20 min. The
cells were then permeabilized using 0.25% Triton X-100. Blocking was performed
using 5% BSA in TBST for 1 h at room temperature to prevent non-specific
binding of antibodies. This was followed by incubation of the cells overnight
at 4 ºC with antibody against TFAM (1:100 dilution) prepared in blocking
buffer. The primary antibodies were then incubated with Alexa Fluor conjugated
secondary antibodies (1:500) for 1hr at room temperature. Cells were washed
thrice with PBS following each step. The cells were finally mounted on a clean
glass slide using VECTASHIELD mounting medium containing DAPI and the images
were acquired using confocal microscope.
staining, cells were incubated with media containing mitotracker (final
concentration: 50nM) at 37 ºC for 30min. After this, cells were washed thrice
with PBS, fixed and permeabilized using methanol at -20 ºC for 20min and
mounted on glass slides.