Supplementary Materials for

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: dhhwang@postech.ac.kr (D.H.); kspark@snu.ac.kr (K.S.P.); bhlee@gachon.ac.kr (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.

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.

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 C I, C II, C III, C IV, and C V. 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.

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 (FDR<0.05 and fold change>1.46; Materials and Methods) from the three comparisons (H versus W, M versus W, and M versus H) were indicated by the asterisk.

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.

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 (H 2 O 2 ; 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 <0.05 by one-way ANOVA with Bonferroni correction. The protein abundance data are representative of three independent experiments. The efficiency of JNK inhibition was shown. The abundance of phosphorylated c-jun (p-c-jun) was used as a marker of JNK activitiy; n = 3, independent experiments. (D) Effects of OXPHOS inhibitors on RXRA abundance. Western blotting analysis of RXRA in W cells after the treatment of dimethyl sulfoxide (DMSO), 3 µm rotenone (R), 15 µm antimycin A (AM), or 12 µm oligomycin (OM) for 18 h; Data are representative of three independent experiments shown in Fig. 5D.

Table S1. Mitochondria-to-nucleus retrograde signaling mediators, signaling pathways, and their downstream TFs. Signaling mediators Signaling molecules / pathways Downstream TFs [Ca 2+ ] c calcinuerin NFATc Effect on the downstream TFs Dephosphorylated (activating their nuclear translocation), increased protein abundance Reference [Ca 2+ ] c Calcinuerin RelA Reduced protein abundance (40) [Ca 2+ ] c Calcinuerin NFkB Increased protein abundance, Increased protein activity (through inactivation of IkBß) (41) [Ca 2+ ] c CaMKIV CREB Increased phosphorylation (42) [Ca 2+ ] c CaMKIV p53 Increased protein activity (42) [Ca 2+ ERK1, ] c ERK2 EGR1 Increased mrna abundance (43) [Ca 2+ ] c JNK ATF2 [Ca 2+ ] c [Ca 2+ ] c JNK and MAPK JNK and MAPK Increased phosphorylation increased protein abundance (40) (40) CEBP Increased protein activity (6, 40, 44) CHOP Increased protein activity (4, 6, 44) [Ca 2+ MEF2, ] c CaMK TORCs Increased protein activity (3, 45) [Ca 2+ ] c CaMKIV PGC1A Increased mrna abundance (46) ROS ERK and Increased mrna expression and EGR1 JNK 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 Increased protein activity p53 p38 (increased phosphorylation) (52) Increased protein activity ROS JNK FOXO (increased phosphorylation), Increased mrna abundance (53, 54, 55, 56) ROS Src HIF1A Increased mrna abundance (57) ROS ROS AMP PI3K and AKT PI3K and AKT AMPK HIF1A NRF1, NRF2 PPARA, PGC1A Increased mrna expression and protein abundance Increased mrna abundance, Increased protein activity (increased phosphorylation) (58) (1, 59, 60, 61) 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)

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

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. Number of interactions Number of TFs TRED BIND AMADEUS EEDB MSigDB MetaCore 7558 9880 12851 41981 154171 13011 111 171 26 179 286 684 Reference (68) (69) (70) (71) (72) Web site http://rulai.cs hl.edu/cgibin/tred/tre d.cgi?process =home http://bind.ca http://acgt.c s.tau.ac.il/a madeus/ http://fanto m.gsc.riken. jp/4/ http://www.b roadinstitute. org/gsea/msi gdb/index.jsp GeneGo, St. Joseph, MI, USA http://www.g enego.com/m etacore.php

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

Table S12. Primer sequences used in qrt-pcr analysis. Sequences are listed 5 to 3. Gene NDUFA1 NDUFA3 NDUFA4 NDUFA7 NDUFA11 NDUFA12 NDUFA13 NDUFAF2 NDUFAF4 NDUFB3 NDUFB7 NDUFB11 NDUFS8 NDUFC1 UQCRB UCRC COX6A1 Primer sequence TGGGCGTGTGCTTGTTGAT CCCGTTAGTGAACCTGTGGATGT CAAGGCCACGCCCTACAAC TCGGGCATGTTCCCATCAT ATGCTCCGCCAGATCATCG GCAAGAGATACAGTGTTGCTCCA CTGTGCCCCCTTCCATCAT TCTGCTGGCTTGCCTGACA CAATCCTCCGGGCACCTT TGCAGTGAACGTGTATTGTCCAA GGCATCGTTGGCTTCACAGT TTACGAGCAGTAAGTGGTTTTGTTGT CGCGCTGTTGCCACTGTTA CCCGAAGCATCTGCAAGGT TGGGTTGGTCTCAGGATTTGTT TGCTCCTTCACTTCCCTTGACA TTATGAGGAGATGGGAGCACTAGTG CCGCTCGGTTCTCTAGGTTGA GCTGGCTGCAAAAGGGCTA CTCCTACAGCTACCACAAATGC ATGTGATGCGCATGAAGGAGTT CTTCTCCCGCCGCTTCTT TCCTTGGCAGCACCTTTGTG CGGCGGGACCACTCTTTC GGCTGAGCCAAGAGCTGATG GATGCACTTGGTCATGTCGATGT GGCCCTTCAGTGCGATCA CCAGTCAGGTTTGGCATTCG ACTGGGGTTAATGCGAGATG GTCCAGTGCCCTCTTAATGC GTGGGCGTCATGTTCTTCGA ACAGCTTCCCCTCGTTGATGT ATGGCGGTAGTTGGTGTGTC GTGAGAGTCTTCCACATGCGA

COX6C CAAAGAAAGAAGGCATACGCAGAT CTGAAAGATACCAGCCTTCCTCAT COX7B CTTGGTCAAAAGCGCACTAAATC CTATTCCGACTTGTGTTGCTACA ATP5D TTTGTGAGCAGCGGTTCCAT GGCCTTCTCCAAGTTTGCCT ATP5L AAGAAATTGAGCGGCATAAG GGAAGCACACAGGTTGATTT MRPS21 GCAAAACATCTGAAGTTCATCGC AGCCCATCCATAGTGAGGATTC MRPL2 ACTCTAACCACATAGGCCGAA TCACTTTCCACGTTGTTGATGAG MRPL51 TTCGAGGTTGGAAAGGGAATG GGATGCGTTTATTAAGGTTGTGC RPL36 ATGGCCCTACGCTACCCTATG CGCACGAACTTGGTGTGTT RRBP1 GGGTTGTGGTCTTTGGAGGAT GGTTGGCTAGGGCTTCTTCATA GAPDH GGTGAAGGTCGGAGTCAACGGA GAGGGATCTCGCTCCTGAAGA

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.