Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin.
ABSTRACT Huntington's disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by an expanded trinucleotide (CAG) repeat in exon 1 of the huntingtin gene (Htt). This expansion creates a toxic polyglutamine tract in the huntingtin protein (HTT). Currently, there is no treatment for either the progression or prevention of the disease. RNA interference (RNAi) technology has shown promise in transgenic mouse models of HD by reducing expression of mutant HTT and slowing disease progression. The advancement of RNAi therapies to human clinical trials is hampered by problems delivering RNAi to affected neurons in a robust and sustainable manner. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and numerous ongoing clinical trials. MSC exhibit a number of innate therapeutic effects, such as immune system modulation, homing to injury, and cytokine release into damaged microenvironments. The ability of MSC to transfer larger molecules and even organelles suggested their potential usefulness as delivery vehicles for therapeutic RNA inhibition. In a series of model systems we have found evidence that MSC can transfer RNAi targeting both reporter genes and mutant huntingtin in neural cell lines. MSC expressing shRNA antisense to GFP were found to decrease expression of GFP in SH-SY5Y cells after co-culture when assayed by flow cytometry. Additionally MSC expressing shRNA antisense to HTT were able to decrease levels of mutant HTT expressed in both U87 and SH-SY5Y target cells when assayed by Western blot and densitometry. These results are encouraging for expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntington's disease.
- SourceAvailable from: Deborah Palliser[show abstract] [hide abstract]
ABSTRACT: As soon as RNA interference (RNAi) was found to work in mammalian cells, research quickly focused on harnessing this powerful endogenous and specific mechanism of gene silencing for human therapy. RNAi uses small RNAs, less than 30 nucleotides in length, to suppress expression of genes with complementary sequences. Two strategies can introduce small RNAs into the cytoplasm of cells, where they are active - a drug approach where double-stranded RNAs are administered in complexes designed for intracellular delivery and a gene therapy approach to express precursor RNAs from viral vectors. Phase I clinical studies have already begun to test the therapeutic potential of small RNA drugs that silence disease-related genes by RNAi. This review will discuss progress in developing and testing small RNAi-based drugs and potential obstacles.Gene Therapy 04/2006; 13(6):541-52. · 4.32 Impact Factor
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ABSTRACT: Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a poly-glutamine expansion in huntingtin, the protein encoded by the HD gene. PolyQ-expanded huntingtin is toxic to neurons, especially the medium spiny neurons of the striatum. At the same time, wild-type huntingtin has important - indeed essential - protective functions. Any effective molecular therapy must preserve the expression of wild-type huntingtin, while silencing the mutant allele. We hypothesized that an appropriate siRNA molecule would display the requisite specificity and efficacy. As RNA interference is incapable of distinguishing among alleles with varying numbers of CAG (glutamine) codons, another strategy is needed. We used HD fibroblasts in which the pathogenic mutation is linked to a polymorphic site: the Delta2642 deletion of one of four tandem GAG triplets. We silenced expression of the harmful Delta2642-marked polyQ-expanded huntingtin without compromising synthesis of its wild-type counterpart. Following this success in HD fibroblasts, we obtained similar results with neuroblastoma cells expressing both wild-type and mutant HD genes. As opposed to the effect of depleting wild-type huntingtin, specifically silencing the mutant species actually lowered caspase-3 activation and protected HD cells under stress conditions. These findings have therapeutic implications not only for HD, but also for other autosomal dominant diseases. This approach has great promise: it may lead to personalized genetic therapy, a holy grail in contemporary medicine.Journal of Neurochemistry 02/2009; 108(1):82-90. · 3.97 Impact Factor
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ABSTRACT: Marrow stem cell regulation represents a complex and flexible system. It has been assumed that the system was intrinsically hierarchical in nature, but recent data has indicated that at the progenitor/stem cell level the system may represent a continuum with reversible alterations in phenotype occurring as the stem cells transit cell cycle. Short and long-term engraftment, in vivo and in vitro differentiation, gene expression, and progenitor numbers have all been found to vary reversibly with cell cycle. In essence, the stem cells appear to show variable potential, probably based on transcription factor access, as they proceed through cell cycle. Another critical component of the stem cell regulation is the microenvironment, so-called niches. We propose that there are not just several unique niche cells, but a wide variety of niche cells which continually change phenotype to appropriately interact with the continuum of stem cell phenotypes. A third component of the regulatory system is microvesicle transfer of genetic information between cells. We have shown that marrow cells can express the genetic phenotype of pulmonary epithelial cells after microvesicle transfer from lung to marrow cells. Similar transfers of tissue specific mRNA occur between liver, brain, and heart to marrow cells. Thus, there would appear to be a continuous genetic modulation of cells through microvesicle transfer between cells. We propose that there is an interactive triangulated Venn diagram with continuously changing stem cells interacting with continuously changing areas of influence, both being modulated by transfer of genetic information by microvesicles.Stem cell reviews 10/2008; 4(3):137-47. · 5.08 Impact Factor
Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's
disease affected neuronal cells for reduction of huntingtin
Scott D. Olson, Amal Kambal, Kari Pollock, Gaela-Marie Mitchell, Heather Stewart, Stefanos Kalomoiris,
Whitney Cary, Catherine Nacey, Karen Pepper, Jan A. Nolta⁎
Institute for Regenerative Cures, University of California Davis Health System, 2921 Stockton Blvd Room #1300, Sacramento, CA 95817, USA
a b s t r a c t a r t i c l e i n f o
Received 25 July 2011
Revised 27 November 2011
Accepted 1 December 2011
Available online 8 December 2011
Mesenchymal stem cell
Huntington's disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by an expanded
trinucleotide (CAG) repeat in exon 1 of the huntingtin gene (Htt). This expansion creates a toxic polyglutamine
tract in the huntingtin protein (HTT).Currently, there is no treatment for either the progression or prevention of
the disease. RNA interference (RNAi) technology has shown promise in transgenic mouse models of HD by
reducing expression of mutant HTT and slowing disease progression. The advancement of RNAi therapies to
human clinical trials is hampered by problems delivering RNAi to affected neurons in a robust and sustainable
manner. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and
numerous ongoing clinical trials. MSC exhibit a number of innate therapeutic effects, such as immune system
modulation, homing to injury, and cytokine release into damaged microenvironments. The ability of MSC to
transfer larger molecules and even organelles suggested their potential usefulness as delivery vehicles for
therapeutic RNA inhibition. In a series of model systems we have found evidence that MSC can transfer RNAi
targeting both reporter genes and mutant huntingtin in neural cell lines. MSC expressing shRNA antisense to
GFP were found to decrease expression of GFP in SH-SY5Y cells after co-culture when assayed by flow cytometry.
U87 and SH-SY5Y target cells when assayed by Western blot and densitometry. These results are encouraging for
expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntington's disease.
© 2011 Elsevier Inc. All rights reserved.
Huntington's disease (HD) is a dominant neurodegenerative
disease caused by polyglutamine repeat expansions in exon 1 of the
huntingtin protein (HTT), which leads to a toxic gain of function
(Huntington's Disease Collaborative Research Group, 1993). HD
characteristics include neuronal inclusions, striatal and cortical
neurodegeneration, chorea, and cognitive and behavioral changes
(Ross and Tabrizi, 2011; Shannon, 2011). The expansion occurs as a
mutation of a naturally occurring trinucleotide (CAG) repeat in exon 1
of the huntingtin gene, also known as IT15, normally encoding a
350-kDa protein (Huntington's Disease Collaborative Research Group,
1993). Htt alleles containing more than 35 CAG repeats generally
cause HD. The disease usually develops in midlife, but juvenile-onset
cases can occur with CAG repeat length over 60 (Langbehn et al.,
2010). Current treatments for HD are limited to managing behavior
and chorea. There are no existing therapies that prevent the death of
striatal neurons or improve the long-term clinical outcome of affected
RNA interference (RNAi) technology employs the use of short
pieces of RNA that are complementary anti-sense to specific regions
of mRNA. The RNAi, when introduced intracellularly, complex with
the RNA Induced Silencing Complex (RISC) and subsequently bind
to the targeted mRNA. The double stranded RNA is then destroyed,
effectively silencing the expression of the gene. RNAi targeting
mutant HTT has proven effective in transgenic mouse models of HD,
where the RNAi expression can easily be turned on in selected tissues
(Boudreau et al., 2009; DiFiglia et al., 2007; Harper et al., 2005;
Maxwell, 2009). Viral vector delivery of short hairpin RNAs (shRNA)
has also been performed in mouse models, and has shown reduced
Molecular and Cellular Neuroscience 49 (2012) 271–281
Abbreviations: MSC, Mesenchymal stem cells; HD, Huntington's disease; HTT,
huntingtin protein; HTT142, huntingtin fragment containing exon 1 and containing
approximately 100 CAG repeats; U87, glioblastoma cell line; SH-SY5Y, neuroblastoma
cell line; GFP, enhanced green fluorescence protein; HTT142gfp, lentiviral vector con-
taining both HTT142 and a GFP reporter; 4dGFP, destabilized EGFP variant with 4 h
half-life; shRNA, short hairpin RNA; shHTT, shRNA antisense to HTT exon 1; shGFP,
shRNA antisense to GFP; shSCRAM, shRNA of same composition of shHTT with several
minor alterations; dsRed-mito, red fluorescence protein reporter with a mitochondrial lo-
calization sequence; ES, embryonic stem cells.
⁎ Corresponding author at: Institute for Regenerative Cures, University of California
Davis Health System, 2921 Stockton Blvd Room #1300, Sacramento, CA 95817, USA.
Fax: +1 916 703 9310.
E-mail address: email@example.com (J.A. Nolta).
1044-7431/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
Molecular and Cellular Neuroscience
journal homepage: www.elsevier.com/locate/ymcne
neuropathy and motor deficits (DiFiglia et al., 2007). However, RNAi
therapy is difficult to translate into human clinical trials due to poor
uptake and transient effects of RNAi when delivered systemically
(Dykxhoorn et al., 2006). The use of RNAi presents a difficulty in
that extracellular RNA has a very short half-life and is rapidly cleared
and degraded in the body. Additionally, as a charged molecule it has
difficulty crossing both the blood brain barrier and cell membranes
(Boudreau et al., 2011; Davidson and McCray, 2011).
Previously only described in plants, (Kehr and Buhtz, 2008;
Waigmann and Zambryski, 1994) recent studies have found that
nucleic acid structures and RNA species can be passed from a
mammalian donor cells to target cells through gap junctions,
exosomes, virtosomes, or tunneling nanotubules (Gahan and Stroun,
2010; Gerdes and Carvalho, 2008; Simons and Raposo, 2009). Feeder
cells of mesodermal origin engineered to express an siRNA against
GFP were found to affect GFP expression in the ES cells with which
they were co-cultured (Wolvetang et al., 2007). Additionally, RNAi
molecules were found to be secreted in microvesicles, which later
fused with other cells (Bruno et al., 2009; Quesenberry and Aliotta,
2008; Skinner et al., 2009; Taylor and Gercel-Taylor, 2011). Ratajczak
et al. described horizontal transfer of mRNA from cell to cell through
embryonic stem cell-derived microvesicles (Ratajczak et al., 2006).
Virtosomeswere recently described
RNA-lipoprotein which can readily enter other cells where they can
modify the biology of the recipient cells (Gahan and Stroun, 2010).
Tunneling nanotubes have also been described for transferring RNA
species and organelles between cells (Gerdes and Carvalho, 2008).
Therefore, we chose to explore a cell-based platform for producing
and delivering RNA interference moieties targeted to the Huntingtin
mRNA as a potential therapeutic avenue for treating HD.
We chose human MSC to produce siRNA, as we and others have
previously shown them to be excellent in vivo delivery vehicles for
enzymes and proteins (Meyerrose et al., 2008, 2010). MSC can be
easily isolated from the bone marrow or adipose tissue and subse-
quently expanded in culture (Meyerrose et al., 2006, 2010). MSC are
immunoprivileged in that they normally elicit no immune response
when used allogeneically. They are anti-inflammatory and secrete a
broad range of trophic factors, including BDNF, NGF, IGF-1, and others
(reviewed in (Joyce et al., 2010). For these reasons, MSC show
therapeutic potential for the treatment of many neurodegenerative
diseases (Joyce et al., 2010). MSC have been shown to be capable of
transferring mitochondria to damaged cells. Spees and Olson et al
demonstrated that the active transfer of mitochondria from MSCs
could rescue aerobic respiration in mammalian cells with nonfunc-
tional mitochondria (Spees et al., 2006). The ability to easily and
safely genetically engineer adult MSC (Bauer et al., 2008; Joyce et
al., 2010; Meyerrose et al., 2010), as well as the ability of these cells
to directly transfer structures as large as mitochondria to target cells
(Spees et al., 2006), to release microvesicles (Bruno et al., 2009;
Simons and Raposo, 2009) and virtosomes (Gahan and Stroun,
2010), and to form gap junctions with target cells in vitro and in
vivo (Matuskova et al., 2010), made MSC an ideal cellular delivery
system for further examination.
The traditional gene therapy approach, where the neurons would
be directly infected in vivo by live lentiviral or AAV vectors carrying
the ShRNA gene of interest suffer from a number of safety concerns.
Integrating virus can pool at the injection site, superinfecting
neighboring cells and limiting distribution beyond a small area.
Viral integrations can be controlled ex vivo with MSC, and verified
by LAM PCR that there is an average of one to two viral integrants
per MSC genome, as suggested by the FDA for stem cell gene therapy
trials. Using MSCs as the delivery vehicle, a “suicide gene” such as
thymidine kinase can be used to eliminate a graft if anything went
wrong. This would not be possible with vector-mediated delivery
since it would destroy the neuron into which the gene had integrated.
We can also use the natural reparative characteristics of MSCs
tobe complexes of
synergistically, and could potentially use their capacity to migrate to
injured cells to potentially better deliver siRNA.
In this study, we utilized viral vectors to create a model system to
detect the transfer of RNAi molecules between MSC and a targeted
cell (Fig. 1). MSC were transduced to express shRNA antisense to
either GFP or HTT mRNA and a scrambled shHTT shRNA (shSCRAM),
as well as a red fluorescent transduction reporter. The MSC were
then co-cultured with neural target cells expressing GFP or mutant
HTT with a GFP transduction reporter. Measurable decreases in GFP
expression, measured by flow cytometry, and HTT, measured by
densitometry were detected in some of the co-cultures assayed.
These findings support the future possibility of MSC-mediated
delivery of therapeutic RNAi to treat HD.
Characterization of MSC engineered to express shRNA
MSC appeared to be unburdened by either the lentiviral transduction
or the expression of any of the shRNAs. As a precaution, we conducted a
series of assays to ensure that transduction had not fundamentally
altered the cells by comparing shRNA-transduced MSC from different
donors to wild type MSC by growth, capacity to differentiate, and by
MSC from donor 2628 were transduced with shHTT and shSCRAM
vectors at an MOI of 80 at efficiencies of 33.09% and 27.32%, relatively,
as measured by dsRed expression, and plated at initial densities of
1000 cells/cm2for growth analysis. Viable cells were then counted
using an MTT assay on sample cultures over time (Fig. 2A). All cultures
demonstrated logarithmic growth without any significant differences
between shRNA expressing cells and their wild type counterparts.
The capability of transduced MSC to undergo osteogenic and
adipogenic differentiation was also confirmed using MSC from donor
2627 transduced with shHTT, shGFP, and shSCRAM at an MOI of 100.
Differentiation was performed for a total of 17 days. Osteogenic medium
induced the formation of calcified extracellular matrix, which stains by
Alizarin red, indicating successful bone deposition (Fig. 2C, bottom
center). Adipogenic medium induced the accumulation of large lipid
droplets in cells that retained Oil Red O dye, a hallmark of adipocytes.
Lentiviral integration and prolonged time in culture could potentially
introduce the possibility of genomic instability. Therefore, karyotypic
analysis was performed on MSC after transduction with shHTT at an
MOI of 80 with three different donors (Fig. 2B) and results were
compared to both non-transduced MSC controls and normal karyotypes.
All karyotypes were analyzed by a cytogeneticist and no abnormalities
were detected on any of the slides.
These results indicate that lentiviral-transduced and culture-
expanded MSC engineered to express shHTT retained the hallmarks of
mesenchymal stem cells, being capable of extended self-renewal and
differentiation into multiple cell lineages. Additionally, the genetic
manipulations and expansion did not cause any changes in growth
rate, chromosomal instabilities or anomalies.
Target protein expression decreases following direct transduction with
The effect of the shRNA lentiviral vectors on the expression of HTT
and 4dGFP was analyzed using flow cytometry and densitometry.
U87 cells were transduced with a lentiviral vector constitutively
expressing mutant HTT and GFP (U87HTT142gfp). Cultures were
then transduced with shGFP or shHTT and protein expression was
monitored over time.
of GFP was demonstrated to decrease over 6 days when measured by
dsRedexpressionincreases intheFL2 channel(Fig.3B). TheshGFPvector
S.D. Olson et al. / Molecular and Cellular Neuroscience 49 (2012) 271–281
GFP-positive to 12.5% GFP-positive cells on day 6 of the culture (Fig. 3C)
measured bygating on GFP-negative
transduction of U87HTT142gfp had a negligible effect on the number of
To ensure expression of HTT142, cell lysates from U87 and
U87HTT142gfp were collected and subsequently analyzed by Western
blot. Fig. 4A shows a 92 kDa protein band recognized by an anti-HTT
antibody present in the U87HTT142gfp cells and not in control U87
cells. The change in this 92 kDa band was then measured using
densitometry. After transduction with shHTT, mutant HTT expression
quickly decreased, as evidenced by the decreasing intensity of the
92 kDa HTT142 fragment. Levels of HTT142 fell to 80% of its starting
level by day 9, normalized to actin (Fig. 4B). In contrast, the control
shGFP vector had no effect on HTT142 throughout the time course.
Target protein expression is reduced following co-culture with MSC
engineered to produce RNAi
In order to detect the transfer of RNAi between MSC and recipient
cells, a series of co-cultures were performed. Initial experiments
focused on reducing the expression of EGFP as a reporter (not
shown). We hypothesized that the long half-life of EGFP (Indraccolo
et al., 2002) was preventing detection of any reduction in
fluorescence over the time-course of the co-cultures. We began
using a destabilized GFP variant with a half-life of only 4 h (4dGFP)
to ensure that any reduction in mRNA would become rapidly
apparent as a reduction in fluorescence. MSC expressing shRNAs,
targeted to 4dGFP and HTT as a control, and a red fluorescent protein
reporter (dsRed-mito) were co-cultured with SH-SY5Y expressing
4dGFP and fluorescence was assayed by flow cytometry after 4 days
Fig. 1. Overview of co-culture system and vectors. A. MSC and U87 or SH-SY5Y cells were transduced to express shRNAs with a dsRed reporter or HTT142 with a GFP reporter re-
spectively. The cell populations were then co-cultured, allowing the RNAi to transfer from the MSC to the other cell by either direct cell to cell contact or indirect contact, where the
shRNA subsequently reduces protein expression. B. The shRNA expression and target vectors are shown here with viral elements colored dark gray, shRNA expression elements
light blue, constitutive promoters dark blue, fluorescent markers green and red, HTT142 yellow, and WPRE light gray.
S.D. Olson et al. / Molecular and Cellular Neuroscience 49 (2012) 271–281
and 10 days in co-culture (Fig. 5). There are 4 different cell popula-
tions easily discernable by different fluorescence profiles in the dot
plots (Supplementary Fig. S1): non-transduced MSCs and SH-SY5Ys
(bottom left), dsRed-positive GFP-negative shRNA expressing MSC
(top left), dsRed-negative SH-SY5Y expressing moderate levels of
4dGFP (bottom middle), and dsRed-negative SH-SY5Y expressing
high levels of 4dGFP (bottom right). After 4 days, the two
co-cultures were indistinguishable by fluorescence, with 4dGFP
expression in the SH-SY5Ys being almost identical when the MSCs
were gated out regardless of whether the MSC expressed an shRNA
targeting 4dGFP or HTT (Fig. 5B left histogram). In contrast, when
the co-cultures were analyzed after 10 days, the green fluorescence
profile of the SH-SY5Ys had decreased significantly in the shGFP
MSC co-culture as compared to shHTT MSC (Fig. 5B right histogram).
We next expanded our system and altered techniques to better optimize
the co-culture system.
The fluorescence based reporter systems had flaws that were
demanding increasingly complicated systems to overcome. In order
to focus our efforts on HD, we instead measured shRNA-mediated
decreases of lentivirally overexpressed mutant HTT levels directly
by Western blot and densitometry. U87HTT142gfp were co-cultured
six times with MSC expressing either shGFP (as a control) or shHTT.
We found mutant HTT142 expression decreased through day 9 in
the target cells (Fig. 6). The U87HTT142gfp cells were pretreated
with mitomycin C to prevent excessive proliferation. This decrease
was present to some extent in both shHTT MSC and shGFP MSC
co-cultures, however, the target protein HTT142 decreased more
when co-cultured with the shHTT –expressing MSC.
Fig. 2. MSC characterization. A. MSC from donor 2628 were transduced with shSCRAM or shHTT at an MOI of 80 and expanded. Representative transduced and wild type cultures at
passage 4 were then analyzed for proliferation using an MTT assay and cell counts were calculated using a standard curve. The results demonstrate logarithmic growth of all the
cultures, with no single culture exhibiting any significant difference compared to any other. B. Karyotypes were generated from all MSC donors used here both before and after
lentiviral transduction. Shown here is a representative karyotype from MSC donor 2627 transduced with shHTT at an MOI of 80, demonstrating lack of any chromosomal changes.
C. MSC from donor 2627 were transduced with shGFP, shHTT, and shSCRAM at an MOI of 100 and were then differentiated, along with wild type MSC, using osteogenic and adipo-
genic inducing media for 17 days. All cultures demonstrated robust differentiation into adipocytes and osteoblasts. Shown are representative images from MSCshHTT stained with
either Oil Red O (top row) at 200× magnification or Alizarin Red (bottom row). Adipogenic differentiation is indicated by the presence of red lipid-containing vesicles in the cell
(top row, right), while osteogenic differentiation is confirmed by the presence of calcified extracellular matrix stained red by Alizarin Red dye (bottom row, middle). There was no
appreciable accumulation of lipid or calcified matrix in the non-induced wells (left).
S.D. Olson et al. / Molecular and Cellular Neuroscience 49 (2012) 271–281
Next, SH-SY5YHTT142gfp cells were co-cultured a total of five
times with MSC expressing either shHTT or shSCRAM as a control in
a variety of culture conditions. Two of five co-cultures showed that
target protein expression decreased at day 5 after co-culture with
blots (Fig. 7). These co-cultures were performed in reduced serum (5%)
Fig. 3. ReductioninGFPfromshGFPasmeasuredbyflowcytometry.TotesttheactivityofGFPtargetedshRNA,U87HTT142gfpcellsweretransducedwithshGFPdsRed-mito.Cellcultures
were collected at day 0, 2, 4 and 6 and were assayed by flow cytometry. A. The green fluorescence of each culture is shown in this histogram overlay. Reduced GFP expression is evident
seen on day 6 (red). B. Overlaid dot plots of the four time points reveals the progressive decrease in green fluorescence (x-axis) while expression of dsRed increases (y-axis) causing the
populations to shift from bottom right to upper left of the plot. C. When compared to no shRNA (green triangle), and shHTT (red square), shGFP (blue diamond)shows a progressive and
dramatic protein reduction. Populations were gated for GFP expression (shown in A) on day 0 and percent GFP-positive was subsequently measured.
Fig. 4. Detection and silencing of HTT142 by shHTT. U87 cells were transduced to express HTT142 and GFP as a transduction reporter. A. Cell lysates from both wild type U87 cells
and U87HTT142gfp cells were collected and proteins were analyzed by Western blot. Anti-HTT antibody revealed the expression of HTT142 as an approximately 92 kDa band pre-
sent only in U87HTT142gfp cells. B. U87HTT142gfp cells were then transduced with either shHTT or shGFP lentivirus, and culture lysates were collected on day 2, 3, 5, and 9 and
compared by Western blot. C. Protein levels were quantified using densitometry and HTT142 expression was normalized to actin. shHTT transduction progressively silenced
HTT142 expression through day 9 (gray diamond), while the control shGFP transduction had no appreciable effect (black square).
S.D. Olson et al. / Molecular and Cellular Neuroscience 49 (2012) 271–281