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SUPPLEMENTARY INFORMATIONdoi:10.1038/nature21408
Table S3. Cell lines, media and source
Cell line Media Source HME MEGM (Lonza) HMEC-Clonetics HMLER MEGM (Lonza) HMEC-Clonetics MCF10A DME+F12 (1:1), 5% Horse
Serum+EGF+Hydrocortisone+Cholera Toxin+Insulin
Dr. Joan Brugge (Harvard Medical School)
BJ-1 DME:Medium199 (3:1)+15%IFS ATCC HUVEC EBM+0.1% hEGF+0.1%
hydrocortisone, 2% FBS, 0.4% bovine brain extract (BBE), 30 μg/ml Gentamicin+15 ng/ml Amphotericin
ATCC
BPLER WIT Media (Stemgent) Dr. Tan Ince (University of Miami)
MCF7ras DME 10%IFS ATCC CAMA1 DME 10%IFS ATCC SKBR3 DME 10%IFS ATCC BT20 DME 10%IFS ATCC MDA-MB-436 DME 10%IFS ATCC HCC38 RPMI 10%FBS ATCC BT474 RPMI 10%FBS ATCC MDA-MB-361 RPMI 10%FBS ATCC T47D RPMI 10%FBS ATCC MDA-MB-231 RPMI 10%FBS ATCC MDA-MB-157 RPMI 10%FBS ATCC HS578t RPMI 10%FBS ATCC HCC1806 RPMI 10%FBS ATCC SUM149 F12 5%IFS, Insulin, Hydrocortisone Dr. Steve Ethier
(University of Michigan)
SUM159 F12 5%IFS, Insulin, Hydrocortisone Dr. Steve Ethier SUM1315 F12 5%IFS, Insulin, Hydrocortisone Dr. Steve Ethier C2C12 DME+15%FBS ATCC Human myoblasts Commercial media Cell Applications
SUPPLEMENTARY INFORMATION
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Table S4. Primer sequences
Primer Forward Reverse GAPDH CGTATTGGGCGCCTGGTCAC ATGATGACCCTTTTGGCTCC CDKN1A ATGTCAGAACCGGCTGGGGATG
TCC CGTGGGAAGGTAGAGCTTGGGCAGG
CDKN1B ATGTCAAACGTGCGAGTGTCTAACG
CTCCACCTCTTGCCACTCGTACTTG
CDKN2A AGCAGCATGGAGCCTTCGGCTGACT
ACCACCAGCGTGTCCAGGAAGCCCT
CDKN2B GAGGAGAACAAGGGCATGCCCAGTG
GAGAGTGGCAGGGTCTGCGCAGTTG
CDKN2C ATGGCCGAGCCTTGGGGGAACGAGT
GGAAACCTGCTCTGGCCGCATCATG
RB1 GCAGCAGTTGACCTAGATGAGATGT
CTTTGAGCAACATGGGAGGTGAGAG
RBL1 GAAGCCCTGGACGACTTTACTGCCA
CTCTCTAGCCTTTCTATACGTTCAC
RBL2 GACAGCTACCGCAGCATGAGCGAAA
TGCCATGTCTTCCCACTTCTTCATC
BRCA1 ATGGATTTATCTGCTCTTCGCGTTG
AATCTCGTACTTTCTTGTAGGCTCC
TP53 ATGGAGGAGCCGCAGTCAGATCCTA
TAGCTGCCCTGGTAGGTTTTCTGGG
ATM GCTTATCTGCTGCCGTCAACTAGAA
CTGATTTCCTGCATCTTTTTCTGCC
CAP2 (cloning and RT)
CAGACGAATTCCACCATGGCCAACATGCAGGGACTGGTGGAAA
GTCTGGGATCCTTAGGCCATAATTTCTGCAGGTTCA
LACTB (RT-1)
GTGGTTGGAGTTTCTGTAGATGGAA
AGTAATCTTGTTGTGACAGAAACCT
LACTB (RT-2)
AAACCTGGTAGTCAGTTTTTGTATT
TGTTAGGGACTGGGGTCCACATCAT
LACTB (cloning)
CAGACGAATTCCACCATGTACCGGCTCATGTCAGCAGTGA
GTCTGGGATCCTCAGTCTGATCTGTCTTTATCAAAT
dS-LACTB GTTATGCGAATTGCTATCATCAGCAAAAGTCTCACC
GGTGAGACTTTTGCTGATGATAGCAATTCGCATAAC
d1-97-LACTB
CAGACGGATCCACCATGCCGCCCTGCTCCAGGTGCTTCGCCA
GTCTGGGATCCTCAGTCTGATCTGTCTTTATCAAAT
REEP1 (cloning and RT)
CAGACGAATTCCACCATGGTGTCATGGATCATCTCCAGGC
GTCTGGGATCCCTAGGCGGTGCCTGAGCTGCTAGCG
PDLIM3 (cloning and RT)
CAGACGAATTCCACCATGCCCCAGACGGTGATCCTCCCGG
GTCTGGGATCCTTAAGCTTTGGGATACAGAGTGACC
SMPX (cloning and
CAGACGAATTCCACCATGAATATGTCGAAACAGCCAGTTTCC
GTCTGGGATCCCTACTGTTCAGCTTTGGGGACATAT
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SUPPLEMENTARY INFORMATION RESEARCH
RT) HER2 AGGTGGTGCAGGGAAACCTG CAGGGGTGGTATTGTTCAGC c-MYC CGACGAGACCTTCATCAAAA CTGCTGTCGTTGAGAGGGTA PISD ATGATGTGTCAGTCAGAGGC CACTGACTTGTACAAAGCCA SNAIL AAGATGCACATCCGAAGCCA CTCTTGGTGCTTGTGGAGCA TWIST CTGCCCTCGGACAAGCTGAG CTAGTGGGACGCGGACATGG SLUG GTTTCAGTGCAATTTATGCA TTCTAATGTGTCCTTGAAGC ZEB1 GTTCTGCCAACAGTTGGTTT GCTCAAGACTGTAGTTGATG SIP1 TCTGAAGATGAAGAAGGCTG AGTGAATGAGCCTCAGGTAA E-cadherin TGCCCAGAAAATGAAAAAGG GTGTATGTGGCAATGCGTTC N-cadherin GACAATGCCCCTCAAGTGTT CCATTAAGCCGAGTGATGGT Vimentin GAGAACTTTGCCGTTGAAGC TCCAGCAGCTTCCTGTAGGT Fibronectin GAGATTGGACCTGCAAGCCCA AGTGCAAGTGATGCGTCCGC ZO-1 AGAGCTGCGGCCGCCAAGAGCA
CAG ATACTGGTTCAGGATCAGGACGACT
CK5 CAGCCGGAGCCTCTACAACC CGCCGAAACCAAATCCACTAC CK8 GATGCTGGAGACCAAGTGGAG GCCGCCTAAGGTTGTTGATG
SUPPLEMENTARY INFORMATION
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Table S5. Antibodies, source and dilution
Antibody Source Dilution (application)
LACTB Proteintech Group # 18195-1-AP 1:5 000 (WB) 1:250 (IF)
PISD Proteintech Group # 16401-1-AP 1:1000 (WB) GAPDH Cell Signaling Technology # 2118S 1:5000 (WB) COX4 Cell Signaling Technology # 4850S 1:5000 (WB) β-actin Cell Signaling Technology # 4970P 1:5000 (WB) Mitochondrial marker EMD Millipore # MAB1273 1:200 (IF) KI-67 BD Pharmingen # 550609 1:200 (IF) CCP3* read below Cell Signaling Technology # 9661S 1:250 (IF) α-actinin Sigma Aldrich # A7811 1:250 (IF) phalloidin Invitrogen # A22283 1:100 (IF) CD44-PeCy7 eBioscience # 25-0441-81 1:200 (FACS) CD24-APC BioLegend # 311118 1:50 (FACS) EPCAM-Pacific Blue BioLegend # 324218 1:100 (FACS) PGS1 Proteintech Group # 17149-1-AP 1:1000 (WB) PGP Proteintech Group # 25081-1-AP 1:1000 (WB) E-cadherin Cell signaling # 3195 1:100 (IF) β-catenin BD Biosciences # 610154 1:100 (IF) Cytokeratin 8 Covance # MMS-162P-250 1:1000 (IF) Zeb1 Cell Signaling # 3396 1:100 (IF) Vimentin Cell signaling # 5741 1:100 (IF) Fibronectin BD Biosciences # 610078 1:100 (IF) h-Ras Santa Cruz #sc-520 1:500 (WB) Calnexin Enzo # ADI-SPA-865 1:2000 (WB) Fis1 Proteintech Group # 10956-1-AP 1:1000 (WB) Opa1 Abcam # ab55772 1:500 (WB) Mfn1 Cell Signaling Technology # 14739 1:1000 (WB) Mfn2 Cell Signaling Technology # 11925 1:1000 (WB) OXPHOS Abcam # ab110411 1:1000 (WB) Drp1 Cell Signaling # 8570 1:500 (WB)
* This CCP3 antibody also recognizes other caspase substrates (such as caspase 9 substrates) apart from cleaved caspase 3. This has been reported on the Cell Signaling website and also in a published report (Yun Fan and Andreas Bergmann, Cell Death Differ., 2010), which specifically describes the cross-reactivity of this antibody with other components of apoptotic pathway. Therefore, in our text we refer to this antibody as “Cleaved Caspase” antibody.
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SUPPLEMENTARY INFORMATION RESEARCH
Table S6. Abbreviations
ATM ataxia-telangiectasia mutated protein CAP2 adenylate cyclase-associated protein 2 CD24 cluster of differentiation 24 CD44 cluster of differentiation 44 CDKN1A cyclin-dependent Kinase Inhibitor 1A CDKN1B cyclin-dependent Kinase Inhibitor 1B CDKN2A cyclin-dependent Kinase Inhibitor 2A CDKN2B cyclin-dependent Kinase Inhibitor 2B cDNA complementary DNA CK5 cytokeratin 5 CK8 cytokeratin 8 COX4 cytochrome c oxidase subunit 4 CSC cancer stem cell DAPI 4',6-diamidino-2-phenylindole DMEM dulbecco's modied Eagle's medium DMSO dimethyl sulfoxide DOX doxycycline dsiRNA dicer-substrate RNAs Edu 5-ethynyl-2'-deoxyuridine EPCAM epithelial cellular adhesion molecule FACS fluorescence-activated cell sorting FBS fetal bovine serum GAPDH glyceraldehyde 3-phosphate dehydrogenase H&E hematoxylin and eosin stain HBSS Hanks' Balanced Salt Solution HME human mammary epithelial cells HRP horseradish peroxidase hTERT human telomerase reverse transcriptase IFS heat inactivated fetal serum LACTB beta-lactamase-like LC-MS liquid chromatography-tandem mass spectrometry LPC lysophosphatidylcholine LPE lysophosphatidylethanolamine mRNA messenger RNA MTUS1 mitochondrial tumor suppressor 1 NOD/SCID nonobese diabetic/severe combined immunodeficiency PBS phosphate-buffered saline PDLIM3 PDZ and LIM domain protein 3 PE phosphatidylethanolamine PG phosphatidylglycerol
SUPPLEMENTARY INFORMATION
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PGP phosphatidylglycerophosphatase PGS1 phosphatidylglycerophosphate synthase 1 PISD phosphatidylserine decarboxylase pRB retinoblastoma 1 protein PS phosphatidylserine qRT-PCR quantitative reverse transcription polymerase chain
reaction RBL2 retinoblastoma-like protein 2 REEP1 receptor accessory protein 1 RIPA radioimmunoprecipitation assay buffer RPM revolutions per minute RT-PCR reverse transcription polymerase chain reaction shRNA short hairpin RNA siRNA short interfering RNA SMPX small muscle protein X-linked TP53 tumor protein P53 ZEB1 zinc finger E-Box binding homeobox 1
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SUPPLEMENTARY INFORMATION RESEARCH
38 KDa31 KDa
52 KDa
76 KDa
38 KDa31 KDa
52 KDa
76 KDa
38 KDa31 KDa
52 KDa
LACTB+GAPDHLACTB
GAPDH
A
AC
C
CB
38 KDa52 KDa
76 KDa
B LACTB
GAPDHB
38 KDa31 KDa
52 KDa
Figure 1a
SUPPLEMENTARY INFORMATION
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Figure 5a
ABCDEF
Raw Western blot image for E (PISD) is shown in Extended Data Fig. 8d
F
A
LACTB
CActinPGS1
PGPD
38 KDa31 KDa
52 KDa
38 KDa52 KDa
76 KDa
38 KDa31 KDa
52 KDa
76 KDa
38 KDa52 KDa
76 KDa
COX4B
38 KDa31 KDa
52 KDa
24 KDa17 KDa
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SUPPLEMENTARY INFORMATION RESEARCH
Figure 5d A B
A BPISD PISD
BLACTB
BCOX4
38 KDa
52 KDa
24 KDa17 KDa
ACOX4
ALACTB
SUPPLEMENTARY INFORMATION
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Extended Data Fig. 2fLACTB+GAPDH
38 KDa31 KDa
52 KDa
76 KDa
Extended Data Fig. 4a
A CB
LACTB+GAPDH
38 KDa31 KDa
52 KDa
76 KDaA B C
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SUPPLEMENTARY INFORMATION RESEARCH
Extended Data Fig. 4f,g,h
A CB
AA
B B
C C
c‐myc
H‐Ras
her2
GAPDH
COX4
actin
38 KDa52 KDa
76 KDa
38 KDa31 KDa
52 KDa
38 KDa
52 KDa
24 KDa17 KDa
102 KDa76 KDa
150 KDa225 KDa
38 KDa31 KDa24 KDa17 KDa
38 KDa31 KDa
52 KDa
SUPPLEMENTARY INFORMATION
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Extended Data Fig. 7e
Raw Western blot images for actin, COX4 and LACTB are shown in Source Data Fig 5a above.
F OXPHOS
ABCDE
F
38 KDa
52 KDa
24 KDa17 KDa
Mfn2E
102 KDa76 KDa
150 KDa225 KDa
Mfn1D
102 KDa76 KDa
150 KDa225 KDa
Fis1A
38 KDa
52 KDa
24 KDa17 KDa
C
Opa1
38 KDa31 KDa
52 KDa
B
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Extended Data Fig. 8g
ABC PISD
calnexinLACTB
A
BC
Extended Data Fig. 8f
ABC
A
B C
LACTB
PISD GAPDH
38 KDa31 KDa
52 KDa
38 KDa31 KDa
52 KDa
76 KDa
31 KDa52 KDa
76 KDa
SUPPLEMENTARY INFORMATION
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Extended Data Fig. 9a
ABC
A PISD
38 KDa31 KDa
52 KDa
76 KDa
COX4C
38 KDa
52 KDa
24 KDa17 KDa
B LACTB
Extended Data Fig. 9d
ABC
A PISD
38 KDa31 KDa
52 KDa
C COX4B LACTB
38 KDa52 KDa
24 KDa17 KDa
38 KDa52 KDa
76 KDa
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Extended Data Fig. 9g
A
B
A LACTB+GAPDH
38 KDa52 KDa
76 KDa
PISDB
38 KDa31 KDa
52 KDa
Extended Data Fig. 10dLACTB+actin
38 KDa52 KDa
76 KDa