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www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1
Supplementary Materials for
A Systems Approach for Decoding Mitochondrial Retrograde
Signaling Pathways
Sehyun Chae, Byung Yong Ahn, Kyunghee Byun, Young Min Cho,
Myeong-Hee Yu,
Bonghee Lee,* Daehee Hwang,* Kyong Soo Park*
*To whom correspondence should be addressed. E-mail:
[email protected] (D.H.); [email protected] (K.S.P.);
[email protected] (B.L.)
Published 26 February 2013, Sci. Signal. 6, rs4 (2013)
DOI: 10.1126/scisignal.2003266 This PDF file includes:
Fig. S1. Association of DEGs with cellular processes and genes
that have been previously reported to be affected by the mt3243
mutation. Fig. S2. Differential expression of TFs known to
participate in mitochondrial retrograde signaling. Fig. S3.
Knockdown of RXRA using siRNA. Fig. S4. Regulation of mRNA and
protein abundances of RXRA through JNK activated by ROS. Table S1.
Mitochondria-to-nucleus retrograde signaling mediators, signaling
pathways, and their downstream TFs. Table S2. Organizing the 2404
DEGs into clusters and their differential expression patterns among
the three types of cells. Table S5. TF-target interactions
collected from the six databases. Table S11. Primary antibodies
used for Western blotting analysis and immunofluorescence analysis.
Table S12. Primer sequences used in qRT-PCR analysis. Tables S3,
S4, S6 to S10 legends
Other Supplementary Material for this manuscript includes the
following: (available at
www.sciencesignaling.org/cgi/content/full/6/264/rs4/DC1)
Table S3 (Microsoft Excel format). The lists of the genes
included in the individual clusters. Table S4 (Microsoft Excel
format). GO Biological Processes enriched in the DEGs in the
individual clusters.
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Table S6 (Microsoft Excel format). Seventy-two TFs that could
potentially be involved in retrograde signaling induced by the
mt3243 mutation. Table S7 (Microsoft Excel format). The OXPHOS
genes differentially expressed in cells containing the mt3243
mutation. Table S8 (Microsoft Excel format). One hundred
sixty-three DEGs that showed an increase in mRNA abundances in
3243G M cells by the RA treatment. Table S9 (Microsoft Excel
format). The translation-related genes differentially expressed in
cells containing the mt3243 mutation. Table S10 (Microsoft Excel
format). One hundred eighty TFs that showed altered mRNA abundance
in cells containing the mt3243 mutation.
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Fig. S1. Association of DEGs with cellular processes and genes
that have been previously reported to be affected by the mt3243
mutation. (A) The top illustrates how the mt3243 mutation causes
mitochondrial dysfunction. The oxidative phosphorylation complexes
are indicated as CI, CII, CIII , CIV, and CV. The number of DEGs
involved in each cellular process is denoted in parenthesis. (B)
The table shows the shared 19 genes and the cluster to which they
belong between the 2404 DEGs in our study and the 56 genes
previously reported by Crimi et al. (8) as affected by the mt3243
mutation.
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Fig. S2. Differential expression of TFs known to participate in
mitochondrial retrograde signaling. Gene expression profiling
revealed that mRNA abundances of seven TFs previously associated
with mitochondrial retrograde signaling were altered by the mt3243
mutation. Transcript abundance obtained from microarrays was
normalized using quantile normalization method. The normalized data
are shown as mean values ± SD; n = 3, independent microarray
experiments. For each gene, all the significant differences
(FDR1.46; Materials and Methods) from the three comparisons (H
versus W, M versus W, and M versus H) were indicated by the
asterisk.
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Fig. S3. Knockdown of RXRA using siRNA. The decrease of mRNA
abundance of RXRA in siRNA-treated indicates that the knockdown was
effective. n = 5, independent experiments. Transcript abundance was
normalized to that of GAPDH. Quantified data are shown as mean ± SD
.
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Fig. S4. Regulation of mRNA and protein abundances of RXRA
through JNK activated by ROS. (A) Western blotting analyses of RXRA
in three types of cells in the presence or absence of 5 mM ascorbic
acid (Asc) for 1 h; data shown are representative of three
independent experiments shown in Fig. 5B. (B) Western blotting
analyses of RXRA in three types of cells in the presence or absence
of 10 µM SP600125 for 1 h; data shown are representative of 3
independent experiments shown in Fig. 5C. (C) qRT-PCR (top) and
Western blotting analyses (bottom) of RXRA abundance in W cells in
the presence or absence of ROS (H2O2; 100 µM for 20 min) with or
without the JNK inhibitor (SP600125; 10 µM for 1 h). The mRNA data
are shown as mean values ± SD; n = 5, independent experiments. ∗
P
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Table S1. Mitochondria-to-nucleus retrograde signaling
mediators, signaling pathways, and their downstream TFs.
Signaling mediators
Signaling molecules / pathways
Downstream TFs
Effect on the downstream TFs Reference
[Ca2+]c calcinuerin NFATc Dephosphorylated (activating their
nuclear translocation), increased protein abundance
(40)
[Ca2+]c Calcinuerin RelA Reduced protein abundance (40)
[Ca2+]c Calcinuerin NFkB Increased protein abundance,
Increased protein activity (through inactivation of IkBß)
(41)
[Ca2+]c CaMKIV CREB Increased phosphorylation (42) [Ca2+]c
CaMKIV p53 Increased protein activity (42)
[Ca2+]c ERK1, ERK2
EGR1 Increased mRNA abundance (43)
[Ca2+]c JNK ATF2 Increased phosphorylation
increased protein abundance (40)
[Ca2+]c JNK and MAPK
CEBP Increased protein activity (6, 40, 44)
[Ca2+]c JNK and MAPK
CHOP Increased protein activity (4, 6, 44)
[Ca2+]c CaMK MEF2, TORCs
Increased protein activity (3, 45)
[Ca2+]c CaMKIV PGC1A Increased mRNA abundance (46)
ROS ERK and
JNK EGR1
Increased mRNA expression and protein abundance
(47)
ROS MAPK AP1 Increased mRNA abundance, Increased
phosphorylation
(48, 49, 50)
ROS - c-MYC Increased mRNA abundance (50) ROS PKD NF-kB
Increased protein activity (51)
ROS ERK and
p38 p53
Increased protein activity (increased phosphorylation)
(52)
ROS JNK FOXO Increased protein activity
(increased phosphorylation), Increased mRNA abundance
(53, 54, 55, 56)
ROS Src HIF1A Increased mRNA abundance (57)
ROS PI3K and
AKT HIF1A
Increased mRNA expression and protein abundance
(58)
ROS PI3K and
AKT NRF1, NRF2
Increased mRNA abundance, Increased protein activity
(increased phosphorylation) (1, 59, 60, 61)
AMP AMPK PPARA, PGC1A
Increased mRNA abundance (62)
AMP AMPK PGC1A Increased protein activity
(increased phosphorylation) (63)
AMP AMPK FOXO3A Increased protein activity
(increased phosphorylation) (64)
AMP AMPK p53 Increased protein activity (53)
AMP AMPK mTOR Decreased protein activity
(increased phosphorylation of TSC2 and raptor)
(65, 66, 67)
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Table S2. Organizing the 2404 DEGs into clusters and their
differential expression patterns among the three types of cells.
The clusters in bold are the 12 clusters that selected for detailed
analyses. The comparisons are indicated as red, increased abundance
and blue, decreased abundance such that, for example, H/W red means
that the abundance was increased in the H cells relative to
abundance in the W cells. See table S3 for the identity of the
genes in each cluster.
Cluster H/W M/W M/H Number of Genes
1 3
2 90 3 1 4 0
5 69 6 59 7 0 8 0 9 8
10 196 11 378 12 0
13 180 14 244 15 0
16 302 17 564 18 7 19 0 20 0
21 69 22 96 23 0 24 10
25 119 26 9
Total 2404
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Table S5. TF-target interactions collected from the six
databases. A total of 223,665
interactions were identified and used for enrichment of the
genes to TFs.
TRED BIND AMADEUS EEDB MSigDB MetaCore™
Number of interactions
7558 9880 12851 41981 154171 13011
Number of TFs
111 171 26 179 286 684
Reference (68) (69) (70) (71) (72) GeneGo, St. Joseph, MI,
USA
Web site
http://rulai.cshl.edu/cgi-
bin/TRED/tred.cgi?process
=home
http://bind.ca http://acgt.cs.tau.ac.il/a
madeus/
http://fantom.gsc.riken.
jp/4/
http://www.broadinstitute.org/gsea/msigdb/index.jsp
http://www.genego.com/metacore.php
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Table S11. Primary antibodies used for Western blotting analysis
and
immunofluorescence analysis.
Antibody target Isotype Company Catalog number Dillution
concentration
NDUFB8 Mouse IgG MitoSciences #MS105 1:1,000
SDHB Mouse IgG MitoSciences #MS203 1:1,000
UQCR2 Mouse IgG MitoSciences #MS304 1:1,000
COX5B Mouse IgG MitoSciences #MS410 1:1,000
ATP5A1 Mouse IgG MitoSciences #MS507 1:1,000
RXRA Mouse IgG Santa Cruz SC-46659 1:2,000
RXRA Rabbit IgG Santa Cruz SC-553 1:2,000
PGC1α Mouse IgG Santa Cruz SC-13067 1:2,000
p-c-jun Rabbit IgG Cell Signaling #9261 1:1,000
β-actin Mouse IgG Sigma-Aldrich A5316 1:5,000
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Table S12. Primer sequences used in qRT-PCR analysis. Sequences
are listed 5’ to 3’.
Gene Primer sequence
NDUFA1 Forward TGGGCGTGTGCTTGTTGAT
Reverse CCCGTTAGTGAACCTGTGGATGT
NDUFA3 Forward CAAGGCCACGCCCTACAAC
Reverse TCGGGCATGTTCCCATCAT
NDUFA4 Forward ATGCTCCGCCAGATCATCG
Reverse GCAAGAGATACAGTGTTGCTCCA
NDUFA7 Forward CTGTGCCCCCTTCCATCAT
Reverse TCTGCTGGCTTGCCTGACA
NDUFA11 Forward CAATCCTCCGGGCACCTT
Reverse TGCAGTGAACGTGTATTGTCCAA
NDUFA12 Forward GGCATCGTTGGCTTCACAGT
Reverse TTACGAGCAGTAAGTGGTTTTGTTGT
NDUFA13 Forward CGCGCTGTTGCCACTGTTA
Reverse CCCGAAGCATCTGCAAGGT
NDUFAF2 Forward TGGGTTGGTCTCAGGATTTGTT
Reverse TGCTCCTTCACTTCCCTTGACA
NDUFAF4 Forward TTATGAGGAGATGGGAGCACTAGTG
Reverse CCGCTCGGTTCTCTAGGTTGA
NDUFB3 Forward GCTGGCTGCAAAAGGGCTA
Reverse CTCCTACAGCTACCACAAATGC
NDUFB7 Forward ATGTGATGCGCATGAAGGAGTT
Reverse CTTCTCCCGCCGCTTCTT
NDUFB11 Forward TCCTTGGCAGCACCTTTGTG
Reverse CGGCGGGACCACTCTTTC
NDUFS8 Forward GGCTGAGCCAAGAGCTGATG
Reverse GATGCACTTGGTCATGTCGATGT
NDUFC1 Forward GGCCCTTCAGTGCGATCA
Reverse CCAGTCAGGTTTGGCATTCG
UQCRB Forward ACTGGGGTTAATGCGAGATG
Reverse GTCCAGTGCCCTCTTAATGC
UCRC Forward GTGGGCGTCATGTTCTTCGA
Reverse ACAGCTTCCCCTCGTTGATGT
COX6A1 Forward ATGGCGGTAGTTGGTGTGTC
Reverse GTGAGAGTCTTCCACATGCGA
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COX6C Forward CAAAGAAAGAAGGCATACGCAGAT
Reverse CTGAAAGATACCAGCCTTCCTCAT
COX7B Forward CTTGGTCAAAAGCGCACTAAATC
Reverse CTATTCCGACTTGTGTTGCTACA
ATP5D Forward TTTGTGAGCAGCGGTTCCAT
Reverse GGCCTTCTCCAAGTTTGCCT
ATP5L Forward AAGAAATTGAGCGGCATAAG
Reverse GGAAGCACACAGGTTGATTT
MRPS21 Forward GCAAAACATCTGAAGTTCATCGC
Reverse AGCCCATCCATAGTGAGGATTC
MRPL2 Forward ACTCTAACCACATAGGCCGAA
Reverse TCACTTTCCACGTTGTTGATGAG
MRPL51 Forward TTCGAGGTTGGAAAGGGAATG
Reverse GGATGCGTTTATTAAGGTTGTGC
RPL36 Forward ATGGCCCTACGCTACCCTATG
Reverse CGCACGAACTTGGTGTGTT
RRBP1 Forward GGGTTGTGGTCTTTGGAGGAT
Reverse GGTTGGCTAGGGCTTCTTCATA
GAPDH Forward GGTGAAGGTCGGAGTCAACGGA
Reverse GAGGGATCTCGCTCCTGAAGA
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Description of the supplementary tables provided as Excel
Table S3. The lists of the genes included in the individual
clusters.
Table S4. GO Biological Processes enriched in the DEGs in the
individual clusters.
Significantly enriched terms (P < 0.05) are shaded orange.
The enriched terms shaded purple
were included in Fig. 2C.
Table S6. Seventy-two TFs that could potentially be involved in
retrograde signaling induced
by the mt3243 mutation. (A) The numbers of target genes of each
TF and their significance
(FDR; see Materials and Methods) in individual clusters. (B) 72
potential TFs that could be
involved in retrograde signaling induced by the mt3243
mutation.
Table S7. The OXPHOS genes differentially expressed in cells
containing the mt3243
mutation.
Table S8. One hundred sixty-three DEGs that showed an increase
in mRNA abundances in
3243G M cells by the RA treatment.
Table S9. The translation-related genes differentially expressed
in cells containing the mt3243
mutation.
Table S10. One hundred eighty TFs that showed altered mRNA
abundance in cells containing
the mt3243 mutation.