The plant alkaloid conophylline inhibits matrix formation of fibroblasts

28 Pages·2017·1.71 MB·English

by  渡辺 秀人

JBC Papers in Press. Published on October 30, 2018 as Manuscript RA118.005783
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.005783
Conophylline inhibits matrix formation of fibroblasts

The plant alkaloid conophylline inhibits matrix formation of fibroblasts

Takehiko Tezuka1, 5), Akinobu Ota2), Sivasundaram Karnan2), Katsuhiko Matsuura1), Kazuhisa Yokoo3),
Yoshitaka Hosokawa2), Davide Vigetti4), Alberto Passi4), Sonoko Hatano5), Kazuo Umezawa6), and Hideto
Watanabe5)

Department of Pharmacy1), Department of Biochemistry2), Department of Plastic and Reconstructive
Surgery3), Aichi Medical University School of Medicine, Department of Medicine and Surgery,
University of Insubria4), Institute for Molecular Science of Medicine, Aichi Medical University5),
Department of Molecular Target Medicine, Aichi Medical University School of Medicine6)
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Correspondence to: ed
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Hideto Watanabe, M.D., Ph.D. Institute for Molecular Science of Medicine, Aichi Medical University h
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1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan. Tel: [phone], ext. 12088. Fax: ://w
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81-561-63-3532, E-mail: [email protected] .jb
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Key words: extracellular matrix, fibroblast, TGFβ, hyaluronan, collagen, fibrosis, conophylline, vinca g
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alkaloid, antifibrotic, stellate cell t on
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ABSTRACT human foreskin fibroblasts, especially focusing on
Conophylline is a vinca alkaloid from leaves of extracellular matrix (ECM) proteins. A gene
the tropical plant Ervatamia microphylla and has microarray analysis revealed that conophylline
been shown to mimic the effect of the growth and remarkably suppressed expression of the gene for
differentiation factor activin A on pancreatic hyaluronan synthase 2 (HAS2) and of its antisense
progenitor cells. However, whereas activin A RNA, whereas the expression of collagen genes
stimulates fibrosis of pancreatic stellate cells, was unaffected. Of note, immunostaining
conophylline inhibits it, suggesting that this experiments revealed that conophylline
compound may serve as an antifibrotic drug. Here, substantially inhibits incorporation of versican and
we investigated the effects of conophylline on collagens into the ECM in cells treated with
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Conophylline inhibits matrix formation of fibroblasts

transforming growth factor β (TGF-β), which connective tissue in stroma obliterates the
promotes collagen synthesis, but not in cells not architecture and interferes with their function,
treated with TGF-β. Moreover, a protein leading to serious diseases. In addition, some
biosynthesis assay disclosed that conophylline chemical agents used for disease treatment, such
decreases collagen biosynthesis, concomitant with as bleomycin, are known to cause fibrosis as a
a decrease in total protein biosynthesis, indicating side effect (3). Therefore, understanding of the
that conophylline-mediated inhibition of fibrosis mechanisms of fibrosis and its regulation have
is not specific to collagen synthesis. Conophylline been general and important issues in the field of
neither affected TGF-β–induced nuclear clinical medicine.
translocation of SMAD family member 2/3 Conophylline (CNP) is a vinca alkaloid
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(SMAD2/3) nor phosphorylation of SMAD2. extracted from leaves of the tropical n
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However, conophylline substantially inhibited plant Ervatamia microphylla (4). This compound ed
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phosphorylation of extracellular signal–regulated was initially found to mimic the effect of activin h
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kinase 1/2 (ERK1/2), suggesting that conophylline A on the differentiation of pancreatic progenitor ://w
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inhibits HAS2 expression via TGF-β–mediated cells (5). It induces differentiation of pancreatic .jb
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activation of the ERK1/2 pathway. Taken together, progenitor cells into insulin-producing β-cells and rg
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our results indicate that conophylline may be a converts cultured ductal cells to β-cells in vitro (5) g
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useful inhibitor of ECM formation in fibrosis. and in vivo (6). Interestingly, whereas activin A t on
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upregulates the expression of α-smooth muscle ua
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INTRODUCTION actin (αSMA) and collagens of pancreatic stellate , 2
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Fibrosis is the formation of excess fibrous cells (PSC) toward pancreatic fibrosis (7), CNP
connective tissue in an organ or tissue in a suppresses their expression (5). CNP inhibits
reparative or reactive process (1). There, stromal progression of non-alcoholic steatohepatitis by
fibroblasts become activated and inhibiting fibrosis (8). These results suggest that
transdifferentiated into myofibroblasts, and these CNP may serve as an anti-fibrosis drug.
cells synthesize high levels of the extracellular Here, we investigated the effects of CNP on
matrix (ECM) molecules including collagens. In behavior of human foreskin fibroblasts
response to injury including wound healing, (NB1RGB). Our microarray analysis revealed that
fibrosis occurs as scarring (2). When fibrosis CNP remarkably suppressed hyaluronan synthase
occurs in organs such as lung and liver, excessive 2 (HAS2) expression, leading to a decrease in
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Conophylline inhibits matrix formation of fibroblasts

hyaluronan (HA). CNP inhibited collagen assay showed no clear cytotoxicity
biosynthesis by a decrease in total protein (Supplementary Figure S3). These results indicate
synthesis. Further analysis suggested that CNP that CNP inhibits cell proliferation without
inhibits the TGF-mediated pathway especially to cytotoxicity at least up to 0.1µg/mL. Therefore,
ERK1/2 but not the Smad2/3 pathway. we treated cells with CNP at 0.025 or 0.1 µg/mL
in the following experiments.
RESULTS To detect specific molecules whose expression
Initially, we treated growing and confluent is affected by CNP treatment, we performed
NB1RGB fibroblasts with different concentrations microarray analysis. When total 27,958 genes
of CNP and examined its cytotoxicity (Figure 1). were analyzed, 39 genes were upregulated >5-fold
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Under a growing phase, CNP at a concentration of and 53 genes were downregulated <0.2-fold after n
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0.1 µg/mL and higher decreased the cell number, 6 hours of CNP treatment in the presence or ed
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which became apparent as early as day 2 after the absence of TGFβ (Figure 2A). Panther h
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treatment (Figure 1B). At day 5, the cells treated classification analysis (PCA) showed that the 22% ://w
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with 0.1 µg/mL CNP decreased to ~40% that of of CNP-suppressed genes encodes the protein .jb
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non-treated cells (Figure 1C). In contrast, related to heterotrimeric G-protein signaling rg
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treatment with 0.025 µg/mL CNP in growing pathway-Gq alpha and G0 alpha mediated g
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phase showed only 20% inhibition of cell pathway (e.g. BDKRB1, RASGRP1, SSTR1 and t on
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proliferation at day5 (Figure 1C). When cells were BDKRB2) and 11% of them encodes the protein ua
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plated at confluence and further cultured, the related to inflammation signaling pathway (e.g. , 2
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MTT levels slightly increased, indicating that cells IL6 and NFATC4) (Figure 2B). In addition, the
slightly proliferated and piled up. When CNP was PCA by cellular component showed that CNP
added to confluent NB1RGB cells, the cell downregulates expression of membrane-related
number decreased at 0.025 µg/mL and higher genes including HAS1 and HAS2 (Figure 2C).
concentration, which became apparent as early as Furthermore, the PCA by molecular function
day 2 (Figure 1B). At 0.1µg/mL CNP, the decrease showed that CNP downregulates expression of
became obvious at day 5 (Figure 1C). By genes encoded catalytic activity (23%, e.g. DGK1,
observation under a microscope, cells were viable HAS1, HAS2 and NOX4), receptor activity (23%,
and attached on the dishes. In addition, both BDKRB1, IL7R, IL21R and SSTR1) and binding
Trypan Blue-exclusion test and alamarBlue○R activity (39%, ADAMTS4, CXCL12 and NFATC4)
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Conophylline inhibits matrix formation of fibroblasts

(Figure 2D). Accordingly, the raw fluorescence in the conditioned media and a trend, not
intensities of both HAS1 and HAS2, which encode statistically significant (p = 0.3, n=6), of decrease
hyaluronan synthesizing enzymes, significantly in HA deposition, correlated with CNP
decreased after CNP treatment compared with no concentrations, were observed in both
treatment (Figure 2E). These results suggest that TGFβ-treated and non-treated samples (Figure 3E,
CNP downregulates expression of HAS genes in F). Interestingly, TGFβ slightly increased HA
NB1RGB fibroblasts. deposition in 3 days of culture (Figure 3F).
To validate the microarray results, we Formation of pericellular HA coat requires
performed qRT-PCR. The levels of HAS2 HA-binding molecules, including versican, TSG-6,
expression dramatically decreased in 3 hours after pentraxin-3, and Inter--trypsin inhibitor (ITI)
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treatment with CNP at either 0.025 or 0.1 µg/mL (9-11). By immunofluorescence staining, versican n
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(Figure 3A). As HAS2 expression is regulated by expression was substantially upregulated by ed
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its antisense RNA (HAS2 AS), we also examined TGF-treatment. In both treated and non-treated h
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its expression levels, and found its substantial samples, versican was localized along with fibers, ://w
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decreases (Figure 3B). As CNP was initially and was not accumulated in the pericellular coat .jb
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found to mimic the effect of activin A, we (Figure 3G). Whereas CNP had little effect on rg
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examined whether activin A inhibits HAS2 versican deposition of cell culture without TGF gu
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expression or not. Interestingly, activin A showed treatment, it substantially inhibited its deposition t on
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little effect on HAS2 expression, suggesting with TGF treatment (Figure 3G, H). Our ua
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specific inhibition by CNP (Figure 3C). microarray data revealed little effect of CNP on , 2
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By erythrocyte exclusion assay, pericellular HA expression of TSG6 and pentraxin-3, although
coat was observed in both TGFβ- treated and TGF substantially up-regulated expression of
nontreated cells (Figure 3D, left panels, TSG6 and down-regulated that of pentraxin 3
arrowhead). CNP treatment at either concentration (Supplementary Figure S1).
of 0.025 or 0.1 µg/mL abrogated pericellular HA We further mined the transcriptomic data for
coat, in both TGFβ-treated and nontreated cells the effects of CNP on glycolytic pathways genes,
(Figure 3D, middle and right panels). When HA including those for production of UDP-sugar
amounts in the cell lysate/ECM and conditioned precursors. No genes related to HA metabolism,
media after three days of culture were measured except for HAS1 and HAS2, showed over 3-fold
using ELISA, significant decreases of HA levels changes of expression (Supplementary Table S1).
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Conophylline inhibits matrix formation of fibroblasts

Taken together, a decrease in HA secretion in When co-treated with TGF, CNP inhibited them
CNP-treated cells is mainly due to remarkable (Figure 4G). Taken together, these results clearly
reduction in HAS expression. indicate that CNP suppresses incorporation of
As CNP was reported to inhibit fibrosis of collagens into the ECM, which is more
pancreatic tissue (5), we investigated the effects of remarkable when cells are treated with TGF.
CNP on collagen secretion and its incorporation Next, we labeled fibroblasts with [3-H] proline
into the ECM. By immunofluorescence staining, and examined the effects of CNP on biosynthesis
CNP treatment substantially diminished type I of collagens and total proteins. The levels of total
collagen incorporation into the ECM (Figure 4A, protein synthesis decreased by treatment with
upper panels). TGF treatment increased collagen CNP at concentrations of both 0.025 and 0.1
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incorporation into the ECM. Under this condition, µg/mL by ~40%. TGF stimulation up-regulated nlo
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CNP treatment dramatically decreased its protein biosynthesis by 50%, and under such ed
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incorporation (Figure 4A lower panels). conditions, CNP treatment inhibited total protein h
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Quantification of the staining intensity confirmed biosynthesis in a similar manner to that of ://w
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the decreased levels, with statistical significance, un-stimulated cells, i.e., by ~35% and 50% at .jb
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of collagen incorporation observed under the 0.025 and 0.1 µg/mL, respectively (Fig. 4H, left). rg
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microscope (Figure 4B). We also investigated The ratio of collagen per total proteins in g
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expression of type III collagen, known to be non-stimulated cells was ~2% in both CNP treated t on
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involved in fibrosis. Immunofluorescence staining and non-treated cells. That in TGF-stimulated ua
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displayed essentially the same patterns with lower cells was ~6%, which was unaffected by CNP , 2
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staining levels than type I collagen (Figure 4C, D). treatment (Fig. 4H, right). These results suggest
Specificity of immunostaining patterns for that CNP inhibits total protein biosynthesis, and
collagens was confirmed by immunostaining its inhibition is not specific to collagen.
using non-immune IgG in place of primary We further mined the transcriptomic data for
antibodies (Figure 4E). Immunoblot analysis the effects of CNP on collagen gene expression,
confirmed that CNP substantially inhibited type I which confirmed that CNP has no specific effects
collagen incorporation (Figure 4F). By SircolTM on collagen expression (Supplementary Table S2).
assay, CNP at both 0.025 and 0.1 µg/mL did not The fact that CNP inhibited collagen
affect collagen levels in the cell lysate and the incorporation more strongly when the cells were
ECM of cell culture without TGF treatment. treated with TGF (Figure 4) suggests that CNP
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Conophylline inhibits matrix formation of fibroblasts

inhibits TGFβ-mediated pathways. TGFβ with CNP further inhibited the phosphorylation in
facilitates differentiation of fibroblasts toward a dose dependent manner (Figure 6A, second
myofibroblasts that actively synthesize collagens. panel and graph). CNP inhibited phosphorylation
Therefore, we examined whether CNP affects the of p38 MAPK in a dose dependent manner. TGF
TGF-mediated pathway and the differentiation. substantially inhibited it, however, combined
When double-stained for total Smad2/3 and treatment with CNP did not affect it (Figure 6A,
SMA, Smad2/3 was mainly localized in the third panel and graph).
cytoplasm in all the TGF non-treated cells. When To investigate whether RAS-ERK1/2 pathway
treated with TGF, Smad2/3 was translocated to was suppressed by CNP treatment, we analyzed
the nuclei in all the samples, regardless of CNP the microarray data, using the GSEA software
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treatment. Without TGFβ treatment, CNP did not program, v2.2.4, and Molecular Signatures n
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affect the number of SMA-positive cells. When Database (Broad Institute). The representative ed
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treated with TGFβ, the number of SMA-positive GSEA enrichment plot and corresponding h
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cells increased at 24 h, which decreased by CNP heatmap image of the indicated gene set for the ://w
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treatment (Figure 5A, B). These results indicate CNP-treated and untreated cells confirmed .jb
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that CNP inhibits TGFβ-mediated differentiation suppression of RAS-ERK1/2 pathway by CNP rg
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of fibroblasts toward myofibroblasts. treatment (Figure 6B). g
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The results shown in Figure 5 suggested that To examine involvement of PI3K/AKT t on
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CNP has little effect on Smad2/3 signal pathway is involved in CNP effects, we analyzed ua
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transduction. When analyzed by western blot, the transcriptomic data, and found that CNP , 2
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TGF-treatment substantially increased slightly inhibits expression of AKT2 and AKT3,
phophoSmad2, and CNP at both 0.025 and but not AKT1 (Supplementary Table S3). When
0.1µg/mL had little effect on phosphorylation of expression of four representative genes
Smad2 (Figure 6A, upper panel and graph), downstream of PI3K/AKT pathway was examined,
confirming the results of Smad2/3 nuclear we observed no remarkable changes by CNP
translocation (Figure 5A). Next, we investigated treatment (Supplementary Figure S2). Western
other signaling pathways mediated by TGF. CNP blot analysis demonstrated little effect on
inhibited phosphorylation of ERK1/2 at both phosphorylation of Akt by CNP (Figure 6A, lower
0.025 and 0.1µg/mL. TGF treatment panel and graph).
substantially inhibited it, and combined treatment
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Conophylline inhibits matrix formation of fibroblasts

DISCUSSION receptors (GPCRs) are known to be major
In this study, we investigated the effects of molecules that regulate HAS2 expression. EGFs
CNP, vinca alkaloid that mimics activin A increase HAS2 expression (13). Early growth
function, on fibroblast behavior and formation of response-1 (EGR-1) induces CD44v6, which then
the ECM. The interesting features of this study sustains ERK signaling, upregulating HAS2
were as follows: (a) CNP dramatically inhibits expression (15). GPCRs act via protein kinase A
HAS 1, HAS2, and HAS2 AS expression, resulting and its downstream CREB1 binds to its response
in a decrease in HA secretion. (b) CNP inhibits elements in HAS2 promoter, inducing HAS2
cell proliferation and biosynthesis of collagens at transcription (16-19). We have shown that CNP
the same ratio with total protein biosynthesis, (c) inhibits ERK1/2 phosphorylation. Our microarray
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CNP inhibits versican incorporation into the ECM, data using the GSEA software program confirmed n
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(d) CNP inhibits TGFβ-mediated differentiation suppression of RAS-ERK1/2 pathway by CNP. G ed
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of fibroblasts toward myofibroblasts, (e) whereas protein-coupled receptors (GPCRs)/protein kinase h
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CNP does not affect Smad2/3 signaling, it inhibits A/CREB1 cascade upregulates HAS2 ://w
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ERK1/2 activation, which may be responsible for transcription (19). Our gene ontology analysis of .jb
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CNP action. Taken together, these findings microarray data shows CNP profoundly inhibits rg
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compellingly indicate the inhibitory effects of the G protein-mediated pathways (Figure 2B). g
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CNP on ECM formation by fibroblasts. Therefore, it is likely that CNP inhibits both t on
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Our microarray analysis revealed marked EGF-mediated and GPCR-mediated pathways, ua
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decreases in HAS1 and HAS2 expression by CNP leading to downregulation of HAS2 transcription. , 2
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treatment among ECM related genes. In addition, Although PI3K/AKT pathway has been reported
the HAS2AS was substantially reduced by the to upregulate HAS2 expression (14), our
treatment, which was validated by qRT-PCR. The microarray analysis did not suggest involvement
measurement of HA in cell lysate and the ECM, of this pathway (Supplementary Figure S2,
and erythrocyte exclusion assay confirmed its Supplementary Table S3). HAS2AS1 forms a
decreased levels by CNP treatment. HAS2 duplex with HAS2 mRNA, and stabilizes the
transcription is regulated by a variety of signal HAS2 transcript (20). In addition, HAS2AS1
transduction pathways involving as many binds to O-GlcNAcylated p65 and induces HAS2
molecules (12-14). Among them, both transcription (12, 21). Reduced HAS2AS levels
EGF-growth family factors and G protein-coupled by CNP treatment may prompt inhibition of HAS2
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Conophylline inhibits matrix formation of fibroblasts

transcription (22-25). accumulated in pericellular matrix.
Incorporation of HA into the ECM involves Whereas CNP was reported to inhibit fibrosis of
HA-binding ECM molecules, such as versican. pancreas (7), our microarray analysis showed no
We have shown that TGF substantially increases significant differences in mRNA levels of
versican expression and CNP inhibits its collagens. Our biosynthesis assay revealed that
incorporation into the ECM, without affecting its TGF upregulates biosynthesis of total proteins
transcription. Whereas versican deposition including collagens and that CNP inhibits protein
decreased to ~60% by CNP treatment, the HA biosynthesis both in the presence and absence of
amount in the cells/ECM and the conditioned TGF (Fig. 5A). Although TGF treatment
medium decreased only slightly. This is consistent increased the ratio of collagens per total proteins,
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with our previous results that mouse embryonic CNP had little effect on the ratio, which agrees n
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fibroblasts (MEFs) with ~20% expression levels with our microarray results. ed
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of versican exhibit ~85% levels of HA in the By both immunostaining and the SircolTM assay, hm
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ECM (26). These observations suggest that the inhibition of collagen deposition by CNP was ://w
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versican is not prerequisite for HA deposition. statistically significant only when treated with .jb
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Interestingly, these MEFs exhibit sustained TGF, although there was tendency of inhibition rg
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HA-mediated signaling and attain premature in the absence of TGF. In contrast, when treated gu
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senescence. Similarly, CNP treatment may alter with TGF to facilitate collagen synthesis, CNP t on
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HA-mediated signaling. Pericellular HA coat substantially inhibited its deposition, suggesting ua
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formation involves HA-binding molecules that the inhibitory activity of CNP on collagen , 2
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including TGS6, pentraxin 3 (11), heavy chains levels is mainly exerted via TGF-mediated
of ITI (10), and versican (9). Our microarray pathway. CNP altered collagen fiber structure
analysis revealed that CNP has little influence on from diffuse fine fibers to broader woven fibers
expression of TSG6 and pentraxin 3. Therefore, with more spaces among fibers. A previous study
these molecules are unlikely to affect HA coat showed a similar collagen fiber patterns by
sizes of CNP-treated fibroblasts. Recently, inhibiting HA deposition, using
versican G1 fragment but not full-length versican 4-Methylumbelliferone (4-MU), HA
forms aggregates with HA and heavy chains of oligosaccharide, and hyaluronidase (28).
ITI in the pericellular matrix (27), which agrees Formation of woven collagen fibers by CNP
with our observation that intact versican was not treatment may be due to a decrease in HA
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Conophylline inhibits matrix formation of fibroblasts

deposition. pathways but not the Smad2/3 pathway, which
We demonstrated that CNP did not affect the facilitates COL1A2 transcription. This supports
Smad2/3 signal transduction pathway, but did our observation that CNP did not inhibit collagen
both ERK1/2 and p38MAPK pathways. CNP biosynthesis in a specific manner. CNP was first
inhibited ERK1/2 phosphorylation in both the identified as a compound that shares the receptor
presence and the absence of TGF, whereas CNP with activin A, but interestingly, it inhibits fibrosis
inhibited p38 phosphorylation only in its absence. whereas activin A facilitates it (5). Whereas
These inhibition patterns suggest that the activin A binds to its receptor and induces
inhibitory effects of CNP is operated via ERK1/2 Smad2/3 signal transduction similar to TGFβ
signaling pathways. As ERK1/2 upregulates cell (34-37), CNP had little effect on the Smad2/3
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proliferation (29) and HAS2 gene expression by pathway. The pathway-specific inhibitory effects n
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activating its downstream CREB, the inhibitory of CNP eliminate its competitive inhibition as a ed
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effects of CNP are likely due to downregulation of ligand with activin A and TGFβ. It may modify h
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ERK1/2 pathway. Crosstalk between the receptor complex or inhibit the function of ://w
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TGF-signaling and ERK1/2-signaling has been intervening molecules between TGF receptors .jb
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reported (30, 31). EGF treatment has been and MAPK. rg
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shown to inhibit basal and TGF-induced In this study, we have demonstrated that CNP gu
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expression of type I collagen and SMA (31), inhibits fibroblast proliferation and ECM t on
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contrary to our observations. Two signaling formation, decreasing incorporation of HA, ua
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pathways may interact differently, dependent on versican, and collagens, without cytotoxicity. , 2
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expression of other molecules, cell types and CNP may be used as an inhibitor of ECM
culture conditions. Previously, CNP was reported formation under pathological conditions, where
to inhibit cell proliferation and collagen secretion excess ECM formation is involved in several
via the p38 MAPK pathway in rat pancreatic pathological processes, such as liver and lung
stellate cells (4, 7, 32), and mainly via JNK and fibrosis, keloid, and hypertrophic scar. CNP may
partly via both ERK1/2 and p38 pathways in Lx-2 be a good tool to control these pathological
hepatic stellate cells (33). The effects of CNP may conditions.
be mediated by different MAPK pathways in
various types of fibroblastic cells. EXPERIMENTAL PROCEDURES
Our results indicate that CNP affects MAPK Cell culture
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Conophylline inhibits matrix formation of fibroblasts

Human foreskin fibroblasts NB1RGB were cultured for 16 hours, CNP at each concentration
provided by the RIKEN BRC through the (0.025, 0.1 μg/mL) was added and the cells were
National Bio-Resource Project of the MEXT incubated for 1 hour. TGF (hereafter, final
Japan, and were grown in DMEM containing 10% concentration of 10 ng/mL) was then added, and
fetal bovine serum (FBS) at 37 °C in a 5% CO cells were cultured for 6 hours. After washing
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incubator. with PBS, cells and extracellular matrix were
Fibroblasts up to three passages were used for collected and applied to microarray analysis. We
all the experiments. performed a comprehensive gene expression
MTT assay, alamarBlue○R assay and trypan blue analysis and compared the results among TGF +
exclusion test and -, and CNP + and – groups, using a Human
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NB1RGB cells were plated in 96 wells at Gene Expression 4 x 44K Microarray chip n
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growing and confluent phases and cultured for 48 (G4845A, Agilent Technologies, Santa Clara, CA, ed
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hours. The medium was replaced with new one, USA), which can examine 27,958 genes. For the h
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CNP (final concentrations of 0.025, 0.1, 0.15, 0.1, analysis of the gene expression profiling, total ://w
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0.2, and 0.3 μg/mL) was added and the cells were RNA was isolated using the RNeasy Mini Kit .jb
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cultured for 24, 48, and 120 hours. Then, MTT (Qiagen) according to the manufacturer’s rg
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(Sigma-Aldrich) dye solution was added to each instruction. The quality of the isolated RNA was g
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well and incubated for 4 hours, and absorption at ascertained using a NanoDrop 1000 t on
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570 nm was determined with an automatic ELISA Spectrophotometer (ThermoFisher Scientific). ua
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plate reader (Multiskan; Thermo Electron, Vantaa, The experimental procedure for cDNA microarray , 2
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Finland) (38). NB1RGB cells at 60% confluence analysis was performed according to the
were treated with CNP for days as indicated. manufacturer’s instruction (Agilent Technologies).
AlamarBlue○R assay (Invitrogen) was performed In brief, cDNA synthesis and cRNA labeling with
according to the manufacturer’s instruction. For cyanine 3 (Cy3) dye were performed using the
trypan blue exclusion test, after staining with 5 Agilent Low Input Quick Amp Labeling Kit
mg/mL trypan blue for 1 min, the number of both (Agilent Technologies).
stained and unstained cells was counted under a The Cy3-labeled cRNA was purified,
microscope. fragmented, and hybridized on a Human Gene
Microarray analysis Expression 4 × 44 K Microarray chip containing
Cells were plated at 80% confluence and 43,377 oligonucleotide probes with the Gene
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