Gene expression profile of the skin in the 'hairpoor' (HrHp) mice by microarray analysis
Gene expression profile of the skin in the Hp 'hairpoor' (Hr ) mice by microarray analysis
Bong-Kyu Kim 0
In-Cheol Baek 0
Hwa-Young Lee 0
Jeong-Ki Kim 0
Hae-Hiang Song 1
Sungjoo K Yoon 0
0 Department of Biomedical Sciences, The Catholic University of Korea , 505 Banpo-dong, Seoul 137-701 , Korea
1 Division of Biostatistics and Department of Medical Life science, The Catholic University of Korea , 505 Banpo-dong, Seoul 137-701 , Korea
Background: The transcriptional cofactor, Hairless (HR), acts as one of the key regulators of hair follicle cycling; the loss of function mutations is the cause of the expression of the hairless phenotype in humans and mice. Recently, we reported a new Hr mutant mouse called 'Hairpoor' (Hr Hp). These mutants harbor a gain of the function mutation, T403A, in the Hr gene. This confers the overexpression of HR and HrHp is an animal model of Marie Unna hereditary hypotrichosis in humans. In the present study, the expression profile of HrHp/HrHp skin was investigated using microarray analysis to identify genes whose expression was affected by the overexpression of HR. Results: From 45,282 mouse probes, differential expressions in 43 (>2-fold), 306 (>1.5-fold), and 1861 genes (>1.2fold) in skin from HrHp/HrHp mice were discovered and compared with skin from wild-type mice. Among the 1861 genes with a > 1.2-fold increase in expression, further analysis showed that the expression of eight genes known to have a close relationship with hair follicle development, ascertained by conducting real-time PCR on skin RNA produced during hair follicle morphogenesis (P0-P14), indicated that four genes, Wif1, Casp14, Krt71, and Sfrp1, showed a consistent expression pattern with respect to HR overexpression in vivo. Conclusion: Wif1 and Casp14 were found to be upregulated, whereas Krt71 and Sfrp1 were downregulated in cells overexpressing HR in transient transfection experiments on keratinocytes, suggesting that HR may transcriptionally regulate these genes. Further studies are required to understand the mechanism of this regulation by the HR cofactor.
With a complex and dynamic structure, hair is
generated by hair-producing follicles and has a patterned
cycle of growth and remodeling, which consists of
growth (anagen), regression (catagen), and rest (telogen)
stages. There are many genes involved in mature hair
follicle (HF) regulation .
One of these genes, hairless (Hr), is expressed in skin,
specifically in the suprabasal cell layer of the
interfollicular epidermis and in the lower portion of the HF
epithelium; its expression is dependent on the hair
cycle. Hr encodes a 130 kDa protein (HR), which
contains a zinc finger domain and is localized in the
nucleus , and acts as a transcriptional corepressor
that regulates transcription through directly binding to
the thyroid hormone receptor [3,4], vitamin D receptor
, and retinoic acid-like orphan receptor a .
Various Hr mutant mice have been studied to
understand the function of HR, and most Hr mutant mice are
created by causing the loss of HR function in their cells,
giving them a typical phenotype with a recessive
inheritance mode [7-14]. Microarray analysis of the skin of
Hrtm1Cct/Hrtm1Cct mice has revealed that loss of HR
function results in specific changes in the expression of
epidermal differentiation-associated genes such as
keratin10, loricrin, filaggrin, keratinocyte
differentiationassociated protein (Kdap), and calmodulin 4, among
other such mouse genes . These results suggest that
HR also plays a role in keratinocyte terminal
differentiation through regulation of gene transcription.
The new Hr mutant mouse called ‘hairpoor’ (Hr Hp)
that we reported recently has a phenotype that is
inherited in an autosomal semidominant manner .
Therefore, the heterozygote shows poor hair distribution,
whereas the homozygote displays total alopecia. The
HrHp mouse genome harbors a T-to-A substitution at
position 403 in the noncoding exon 2 of Hr, and this
mutation confers overexpression of HR in the mutant
mouse skin . This clearly distinguishes HrHp mice
from other Hr mutant mice with loss of function of Hr.
Marie Unna hereditary hypotrichosis (MUHH;
OMIM-146550) is an autosomal dominant disorder that
displays coarse and twisted hair development in early
age in humans and progresses to alopecia as the patients
grow older. Recently, the genetic cause of MUHH was
found to be similar to the mutation found in Hr Hp,
namely a mutation in the 5’ UTR of the HR gene
[16,17]. This makes the HrHp mouse one of the valuable
animal models for MUHH, and studying HrHp mice will
facilitate the understanding of MUHH pathogenesis.
HrN mutant is another model for MUHH [15,18]. We
recently reported that overexpression of HR is
associated with alterations in the morphology and
expression of a number of genes in the skin of the HrHp
mouse . In the present study, we performed
microarray analysis and compared HrHp/HrHp skin with that
of age-matched wild-type mice to identify the genes
whose expression was affected by the overexpression of
HR. We catalogued the genes showing differential
expression in the mutant skin and found some of them
to be tightly regulated by HR, which we confirmed
using a reporter expression system.
Hr overexpression preceded histological changes in the
skin of HrHp/HrHp mice
Although we have shown that the overexpression of HR
causes a number of morphological alterations in the
skin, we know little about the molecular basis of these
changes. Because the HR protein functions as a
transcriptional co-repressor [4,5], we set out to identify the
genes whose expression was specifically affected by HR
overexpression using microarray analysis. To investigate
the initial events underlying the morphological changes,
we determined the time point at which morphological
changes and Hr expression in the skin of HrHp/HrHp
mice first occurred.
The fur of +/+ and HrHp/HrHp mice showed
noticeable differences by P7, so we examined the histology of
the skin at the earlier time points by hematoxylin and
eosin staining. At E18.5 and P0, we did not find any
differences in the skins of +/+ and HrHp/HrHp mice (Figure
1A-D) However we observed that HFs were much
shorter and did not grow deep into the subcutis layer in
HrHp/HrHp skin than in the +/+ skin at P3. In addition,
hair bulbs in the HrHp/HrHp mice displayed short and
round shape compared to the extended orval shape of
the hair bulbs in the wiltype mice (Figure 1G). This
observation clearly showed the morphological changes
occurred in the mutant skin at P3.
Next, we compared HR protein expression in the skin
of HrHp/HrHp mice with that of +/+ mice by western
blot analysis. The expression of HR was increased in
HrHp/HrHp skin compared with +/+ skin at P0 and P3,
as shown in Figure 1H, indicating that HR was
overexpressed in HrHp/HrHp skin.
Based on these results, we decided to perform
microarray analysis on the skin of mice at P0 as HR was
overexpressed without prominent morphological changes in
the skin of HrHp/HrHp compared to that of +/+ mice at
Microarray expression profile of genes differentially
expressed in the skin of HrHp/HrHp mice
Using 45,282 mouse probes, we performed microarray
analysis and detected differential expression in 43
(>2fold, p < 0.05), 306 (>1.5-fold, p < 0.05), and 1861 genes
(>1.2-fold, p < 0.05) in the skin of HrHp/HrHp mice
compared with the skin of +/+ mice at P0 (Figure 2,
Table 1), listed in Table 2. Among the 43 genes with >
2-fold expression, 33 were downregulated in HrHp/HrHp
skin at P0. The most strongly downregulated genes in
the mutant skin included Cidea (0.14-fold), Cyp2g1
(0.18-fold) and Krt71 (0.3-fold). Contrasting this
downregulation, the expression of 10 genes, Sepina3h
(2.96fold), Hmgcs2 (2.16-fold), and Odc1 (2.1-fold) in
particular, were significantly upregulated despite HR being a
In addition, we also analyzed microarray data using
q-value. We found differential expression of 23 (>2-fold,
q < 0.05), 90 (>1.5-fold, q < 0.05), and 283 genes
(>1.2fold, q < 0.05) in the skin of HrHp/HrHp mice compared
with the skin of +/+ mice at P0. Fewer genes were
found to be differentially expressed based on the
q-values than on the p-values. While some of the genes
with higher fold change in expression were found to be
not differently expressed (Cox7a1 and Hbb-b1), many
genes such as Cidea, Cyp2g1, Krt71 Sepina3h, Hmgcs2
and Odc1 showed significant differential expression with
significant q-values (Table 2). All of 283 genes are listed
in Additional file 1 and 2.
Using the database of Kyoto Encyclopedia of genes
and genomes (KEGG), 306 genes (>1.5-fold, p < 0.05),
which were identified to be affected by overexpression
of HR in HrHp/HrHp mice, were found to be involved in
the biological pathways related to cell-cell signaling and
communication, various cancers, metabolism, and
regulation of the actin cytoskeleton (Table 3). These results
suggested that pathways involved in the communication
and proliferation of cells were affected by HR
Figure 1 HR was overexpressed without prominent morphological changes in the skin of HrHp/HrHp and +/+ mice. (A-F) Cross-sections
of the skin of hairpoor mice (HrHp) during early stages (E18.5, P0, and P3). At E18.5 (A, B) and P0 (C, D), noticeable differences were not found
between +/+ and HrHp/HrHp skins. At P3 (E, F), There were shorter HFs detected in the HrHp/HrHp skin. HF did not grow deep into the subcutis
layer in HrHp/HrHp skin than in the +/+. Scale bar = 100 μm. (G) Shape of hair bulbs in the +/+ and HrHp/HrHp skins at P3. (H) HRoverexpression in
the skin of HrHp/HrHp mice at P0 and P3. Western blot analysis was performed with protein extracts from the backsides of mice at P0 and P3
using an HR antibody. The a-tubulin indicates equal amount of protein loading.
Expression pattern of HF associated genes during HF
As the first step to delineating the function of the genes
whose expression was affected by HR overexpression,
we assessed genes directly associated with HF
development. Because few genes were directly associated with
HF morphogenesis and/or development in the 43
(>2fold) or 306 (>1.5-fold) genes showing differential
expression, we broadened our search to include the
1861 genes (>1.2-fold). Among those genes, we focused
on the Wnt signaling pathway-associated genes (Sfrp1,
Wif1, Wnt7b), caspase-14 (Casp14), Janus kinase-2
(Jak2), keratins (Krt71, Krt15) and fibroblast growth
factor 10 (Fgf10) (Table 4). Wnt signaling is not only
involved in HF development [19,20] but also in HF
cycling . HR is reported to play a role in these
processes [15,21]. HF undergoes vast apoptosis during
catagen; Casp14  and Jak2  play a role in apoptosis,
and in addition, Casp14 is directly associated with
epidermal cell differentiation . Krt71 and Krt15 are
some of the main constituents of structures that grow
from the skin. Krt71 is expressed in the inner root
sheath (IRS), specifically in Henle’s and Huxley’s layers
, and Krt15 is expressed in the basal layer of the
outer root sheath . Fgf10 is expressed in dermal
papilla, the outer root sheath, and keratinocytes .
We validated the microarray analysis data using
realtime PCR. This was carried out using gene-specific
Figure 2 Gene expression profile in HrHp/HrHp skin using
microarray. (A) Hierarchical clustering represents differential
expression of the genes between +/+ and HrHp/HrHp skins. (B) Using
DEG finding criteria, we found differential expression in 43 (>2-fold)
and 306 genes (>1.5-fold) in the skin from HrHp/HrHp mice
compared with the skin from wild type mice.
primers and the same RNA sources used for the
microarray analysis. Results found by real-time PCR
corroborated those from the microarray analysis, as shown in
Table 4. Six upregulated genes, Wif1, Wnt7b, Casp14,
Jak2, Krt15, and Fgf10, showed similar or higher
upregulation by real-time PCR than microarray analysis. Two
downregulated genes, Sfrp1 and Krt71, showed a similar
fold reduction in expression with both measurement
To analyze the potential role of these genes in HF
morphogenesis, we investigated their expression during
early HF morphogenesis (P0-P14) by comparing their
expression levels in the skin of mutant mice with those
of wild-type mice. Further real-time PCR analysis
revealed that the downregulated genes, Krt71 and Sfrp1,
Table 1 Summary of microarray analysis result
Table 2 Partial list of up-regulated genes and
downregulated genes in the skin of HrHp/HrHp at P0 compared
with that of age matched wild type (>1.5-fold, p < 0.05)
Table 3 KEGG pathway associated with Hr overexpression
maintained their decreased expression status in the
mutant skin throughout the various developmental
stages. The relative expression levels of Krt71 and Sfrp1
mRNA in the mutant skin was decreased to 0.07-fold
and 0.39-fold of those in the wild type skin at P14,
On the other hand, there were two subclasses of the
upregulated genes; one group displayed a consistent
expression pattern, whereas the other group showed an
inconsistent pattern with respect to the HR expression.
The relative expression levels of Wnt7b, Krt15, Jak2,
and Fgf10 did not show a consistent pattern with respect
to the HR expression in HrHp/HrHp mice. In contrast,
the levels of Wif1 and Casp14 mRNA gradually
increased over time. Thus, by P14, the relative
expression levels of Wif1 and Casp14 in HrHp/HrHp skin were
5.77- and 5.35-fold higher than those of +/+ skin,
respectively. This continuous increase in expression was
consistent with the HR overexpression pattern in HrHp/
HrHp mice (Figure 3) .
Expression of Wif1, Casp14, Sfrp1, and Krt71 in
To further analyze whether the expressions of Wif1,
Sfrp1, Casp14, and Krt71 were directly regulated by HR,
we investigated changes in expression of these genes in
the presence of overexpressed HR in a transient
expression system using the mouse keratinocyte cell line,
PAM212. RT-PCR revealed all the genes normally
expressed in PAM212 cells (Figure 4A); transfection of
PAM212 cells with Hr cDNA construct resulted in
expression of HR (Figure 4B). The expression of all four
genes was affected by the presence of HR: expression of
Wif1 and Casp14 was increased 1.85- and 1.57-fold
in HR-overexpressed cells compared to the
mocktransfected PAM212 cells, respectively. In contrast, the
relative expression of Sfrp1 and Krt71 was decreased to
0.61- and 0.52-fold (Figure 4C) in HR-expressing cells
compared with control cells. These results were
consistent with their expression pattern in vivo and strongly
suggested that HR may directly regulate expression of
Recently, we reported the HrHp mouse generated by
N-ethyl-N-nitrosourea mutagenesis as an animal model
of human MUHH . As an initial step to delineate
the molecular basis of the underlying mechanism for the
HrHp phenotype, we investigated the differential
expression of genes in the skin immediately before
morphological changes occurred in the HrHp/HrHp mouse.
Table 4 Validation of the differential expression of the selected genes in the mutant skin
Homozygote/wild type Fold change
Wnt signaling associated factor
Apoptosis associated factor
Figure 3 Differential expression of the genes of interest during
HF development with real-time PCR. Four genes showed a
consistent pattern of expression corresponding to Hroverexpression.
Wif1 and Casp14 were upregulated (A) whereas Sfrp1 and Krt71
were downregulated (B) throughout the developmental stages. The
remaining genes (Wnt7b, Jak2, Krt15, and Fgf10) displayed a
complex expression pattern (C) during developmental stages
(P0P14). The Y-axis indicates the fold difference in expression, showing
the relative expression level of each gene in HrHp/HrHp mice
compared with the skin of age-matched +/+ mice. The values are
the average of the relative expression level of three mice, each
measured in duplicate.
Microarray analysis revealed that various biological
pathways and the expression of many genes were
affected by overexpression of HR.
Other studies have reported systematic screening of
differentially expressed genes in the skin of mice with
the Hr mutation, including analyses of gene expression
in the skin of HrN mice, another Hr-overexpressing
mutant, and Hrtm1Cct, an Hr-loss-of-function mutant
[7,18]. HrN mutants harbor the mutation A402G, which
abolishes the same uATG as in HrHp mutants, indicating
that they have an identical defect . A comparison of
our microarray analysis results with those reported for
the HrN mutant did not show that the same genes
displayed differential expression. This may be due to the
difference in the developmental stage of the HF and
epidermis (P0 vs. P7 or 5 weeks after birth) used in these
analyses. However, many keratin-associated protein
genes were detected in both mutants. At P0 in HrHp/
HrHp mice, the expression of Krtap6-2, Krtap16-7, and
Krtap16-3 increased, whereas the expression of
Krtap51 and Krt71 decreased. Similarly, Krtap6-3, Krtap8-2,
Krtap14, Krt1-1, and Krt1-3 were downregulated in
HrN/HrN mice at P7 . These results suggest that
HR-regulated genes are associated with keratinocyte
differentiation and/or hair-shaft structure. Furthermore,
changes in the expression of keratin10, Kdap, and many
epidermal differentiation-associated genes were also
detected in Hrtm1Cct/Hrtm1Cct mice at P12 . Results
suggest that mutation of Hr causes the abnormal
expression of many keratin-associated genes during HF
morphogenesis and result in disruption of normal hair
Of four genes with consistent expression patterns with
respect to HR overexpression, two genes, Sfrp1 and Wif1,
belong to Wnt inhibitor families. Both inhibitors interfere
with Wnt signaling transduction by binding directly to
the WNT protein [28,29], but though they function in a
similar fashion in the Wnt signaling pathway, they
displayed completely different expression patterns in HrHp/
HrHp skin compared with age-matched +/+ skin.
Surprisingly, Sfrp1 was downregulated by overexpression of the
HR protein, whereas Wif1 was upregulated. Although we
cannot rule out the possibility that this result may be
caused by different locations of Sfrp1 and Wif1
expression in the skin, this difference is more likely to result
from the differential transcriptional regulation of these
genes, as seen in keratinocyte cells with HR
overexpression (Figure 4). The Hr overexpression in keratinocyte
cells results in suppression of Sfrp1 by 39% and activation
of Wif1 by 85%, suggesting that these promoters respond
to HR differently. It is not known whether HR functions
as an activator for Wif1 transcription, and further study
is required to understand its mechanism of action.
Regulation of the Wnt pathway by HR through transcriptional
regulation of Wise, Soggy and Sfrp2 is reported to be
important for proper HF cycling. While expression of
Wise and Soggy mRNAs were upregulated in the
Hrtm1Cct/Hrtm1Cct skin. Their expression levels were
reduced in the skin of HR over-expressing transgenic
mouse (2-fold). Sfrp2 was also shown to be
downregulated in the HrHp/HrHp skin. [16,21,30]. We may include
two more Wnt inhibitors, Sfrp1 and Wif1, for being
regulated by HR based on this work. Further study is required
Figure 4 Changes in expression of Wif1, Sfrp1, Casp14, and Krt71 in Hr-transfected PAM212 cells. (A) Expression of Wif1, Sfrp1, Casp14,
and Krt71in normal PAM212 cells using RT-PCR. M: size marker. (B) Western blot analysis showing the HR protein expressed in Hr-transfected
PAM212 cells. b-tubulin indicates equal amount of protein loading. (C) Regulation of Wif1, Sfrp1, Casp14, and Krt71 gene expression by HR in
Hrtransfected PAM212 cells by real-time PCR. The Y-axis indicates the fold difference in relative expression levels of each gene in HR-overexpressing
cells compared with controls. *p value < 0.05. Results are the average of three independent experiments conducted in duplicate.
to understand their function(s) in HF development and/
Casp14 is another gene upregulated by overexpression
of HR, and is expressed in IRS and corneous cells of the
outer root sheath in HFs . It plays a role in the
formation of the stratum corneum and terminal
differentiation of keratinocytes [22,31]. The higher levels of
Casp14mRNA suggest that an increase in terminal
differentiation of keratinocytes occurs in the mutant skin. This
result is comparable with our observation that the
epidermis of the mutant skin shows increased differentiation in
three-week-old mutant mice compared with age-matched
wild-type mice . Interestingly, Casp14 is also highly
expressed in Hrtm1Cct/Hrtm1Cct mice, which display the
hairless phenotype and show an increase in terminal
differentiation of the epidermis with loss of function of Hr
. Furthermore its striking up-regualtion is age specific,
which closely resembles its expression pattern in the
HrHp/HrHp mice. Thus, despite having the opposite
molecular defect, HrHp/HrHp and Hrtm1Cct/Hrtm1Cct mice
exhibit increased expression of Casp14and displayed a similar
increase in the terminal differentiation of keratinocytes,
suggesting that, Casp14 may play an important role in
proper differentiation of ketatinocyte. Thus, although it is
not clear how HR regulates Casp14expression, both loss
of Hr expression and Hr overexpression lead to alopecia
through expression of Casp14 expression. Krt71 is also
expressed in the IRS, specifically in Henle’s and Huxley’s
layers; gene knockout mice show irregularly formed
Henle’s and Huxley’s layers compared with the wild-type
mice, indicating that Krt71 plays an important role in the
formation of linear IRS intermediate filaments .
Results show that the downregulation of Krt71expression
in the skin of HrHp/HrHp mice is comparable with that in
Krt71 knockout mice in that IRSs in HrHp/HrHp mice
were also abnormal.
Wif1 and Casp14 were found to be upregulated, whereas
Krt71 and Sfrp1 were downregulated by overexpression
of HR. These results suggest that HR may
transcriptionally regulate these genes. Clearly, further studies are
required to delineate the molecular mechanisms
underlying how HR regulates its target genes and to elucidate
the role of Hr in HF development, leading to a better
understanding of MUHH.
Histological study of the skins of wild-type and HrHp/HrHp
The skin was gathered from the buttocks of mice at E
(Embryonic day)18.5, P0(Postnatal day 0) and P3 of
wild-type (+/+) and HrHp/HrHp as previously described
. Paraffin sections were prepared, cut 5-μm in
thickness and stained with hematoxylin and eosin (H&E),
following a standard method. The histological morphology
was observed with an optical microscope (Olympus).
Western blot analysis
Protein extracts were prepared from mouse skin at P0,
and P3 in RIPA buffer (150 mM sodium chloride, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM
Tris-HCl pH8.0) following a standard method. Bradford
assay was performed to quantify protein amount. Two
hundred micrograms of protein were used for western
blot analysis as described earlier . Rabbit polyclonal
HR antibody  and a- tubulin antibody (Santa Cruz)
were used at a dilution of 1: 5000 and 1:2500,
respectively. Antigen-antibody complexes were detected using
ECL system (Amersham Bioscience) and exposure to
x-ray film (Kodak).
Microarray hybridization and data analysis
Total RNA was extracted from the skins of wild type and
HrHp/HrHp mice at P0 (N = 6) using TRIZOL following
the manufacturer’s instructions (Invitrogen). RNA that
passed the quality check using an Agilent Bioanalyzer
were used for microarray analysis using Mouse WG-6
v2.0 Expression BeadChip Kits (Illumina). Total RNA
was reverse transcribed to cDNAs which were
used to synthesize biotin labeled cRNA in an in vitro
transcription reaction (Ambion). Two micrograms of
biotin-labeled cRNA were loaded on to BeadChip and
hybridization and washing experiments were carried out
following the protocol from the manufacturer (Illumina).
The slides were scanned using Illumina BeadArray Reader.
Gene pathway analysis
To analyze biological pathways associated with
differentially expressed genes in the mutant skin, we input a
total of 306 genes (>1.5 fold, p < 0.05) to the KEGG
The ‘affy’ and ‘gcrma’ packages of Bioconductor were
used to preprocess and normalize the data following
import of CEL files into the R statistical package
(Affymetrix, Inc, Santa Clara, CA, USA). The GC Robust
Multiarray Analysis (GC-RMA) was used to adjust
perfect match (PM) probe data for background noise.
Normalization was performed on adjusted perfect match
(PM) data with an algorithm based on rank invariant
After normalization, differential gene expression
between groups was assessed by Significance Analysis of
Microarrays (SAM) . The t-test was calculated for
statistical comparisons, and p-values were obtained with
100 permutations. The q-value, which is a Bayesian
equivalent to the false discovery rate (FDR)-adjusted
p-value, is estimated . The q-value is a well suited
measure of significance for the genomewide tests of
RT-PCR and Realtime PCR
Total RNA was extracted from the skins of wild type
and HrHp/HrHp mice at P0 (N = 6), P3 (N = 3), P7
(N = 3), P10 (N = 3) and P14 (N = 3) as described
above. Single stranded cDNAs were synthesized using
the PrimeScript 1st strand cDNA Synthesis kit (Takara).
PCR was performed using Peltier Thermal Cycler-100
(MJ Research). PCR conditions were 2 min at 95°C
followed by 28 cycles of 15 sec at 94°C, 15 sec at 62°C, 15
sec at 72°C. The final extension was for 10 min at 72°C.
Forward and reverse primer sequences of each gene are
listed in Additional file 3. Realtime PCR was performed
with SYBR Premix Ex Taq (Takara) using an Mx3000P
(Stratagene). The cycling condition was initial
denaturation for 2 min at 95°C followed by 45 cycles of 15 sec at
94°C, 15 sec at 62°C and 15 sec at 72°C. The gene
expression levels were measured by the comparative
ΔΔCt method , and the relative mRNA expression
levels were determined based on the realtime PCR
performed in duplicate using three independent samples.
Statistical significance was determined by a student
ttest using Sigma plot.
Cell culture and transient transfection experiment
Mouse keratinocyte cells (PAM212 cell line) were
cultured in DMEM (Invirogen) containing 10% FBS with
5% CO2 at 37°C incubator. An Hr full-length cDNA
clone (BC049182) and pcDNA 3.1(+) vector were
purchased from Invitrogen. Transfection was carried out
using polyethyleneimine (Sigma-Aldrich) following the
manufacturer’s instruction. 8 x 105 cells were seeded in
60 mm dishes in triplicate. Twenty-four hours later,
3 μg of Hr cDNA construct and 0.2 μg of
pCMV3.1/bgal were introduced into cells, which were harvested 48
hr post transfection and protein and total RNAs were
extracted for western blot and realtime PCR analyses,
respectively. Plasmid pcDNA 3.1 DNA was used as
a control, and the relative expression level was
normalized against transfection efficiency determined by
Additional file 1: Up-regulated genes in the skin of HrHp/HrHp at P0
compared with that of age matched wild type (>1.2-fold, p and q <
Additional file 2: Down-regulated genes in the skin of HrHp/HrHp at
P0 compared with that of age matched wild type (>1.2-fold, p and
q < 0.05).
Additional file 3: List of gene-specific primers.
The Korea Research Foundation Grant funded by the Korean Government
(KRF-2008-313-E00397) and a research grant of the Life Insurance
Philanthropy Foundation supported the study.
The research was conducted under SJKY’s direction. BKK performed all
experiments and analyzed data. ICB and HWL prepared skin sample and
performed histological experiment. JKK and SJKY helped in drafting and
revising manuscript. HSS analyzed the microarray data and calculate p- and
q-values. BKK and SJKY wrote paper. All authors read and approved the final
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