Lclat Agpat Lpcat Agpat Lpcat Lpcat Lpcat Lpgat Awat1 N.D. Mboat Awat2 N.D.
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- Φυλλίς Βενιζέλος
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1 Supplemental Table I. The gene expression of 7 acyltransferases in MOVAS-1 cells. Gene Name (pg)/18s(ng) Gene Name (pg)/18s(ng) Acat Gpat 9.75 Acat. Lclat1 1.5 Agpat Lpcat Agpat.16 Lpcat. Agpat Lpcat Agpat 9.9 Lpcat 9.9 Agpat5 3.3 Lpgat1 9.5 Awat1 N.D. Mboat Awat N.D. Mboat N.D. Dgat1. Mboat7 N.D. Dgat.1 Mogat1 N.D. Gpat Mogat 1.9 Gpat N.D. Sptlc1.1 Gpat3.16 The quantitative expression values of 7 acyltransferase were calculated using the absolute standard curve method of real-time qpcr with plasmid templates containing each full length target gene.
2 Supplemental Table II. shrna Clone IDs used for generating stable knockdown cells Gene Name Clone ID Acat1 Acat Agpat1 Agpat Agpat3 Agpat Agpat5 Atf6 Dgat1 Dgat Gpat1 Gpat3 Gpat Ire1 Lipin Lpcat1 Lpcat Lpcat3 Lpcat Lpgat1 Mboat1 Mogat Scd Sptlc1 VLMM_3791 V3LMM_3666 TRCN17386 VLMM_696 V3LMM_683 V3LMM_6166 V3LMM_1599 V3LMM_5916 TRCN1789 TRCN1576 TRCN1156 TRCN9985 V3LMM_195 V3LMM_599 TRCN988 TRCN183 V3LMM_5893 V3LMM_51111 V3LMM_519 TRCN1569 V3LMM_5116 TRCN18 V3LMM_51655 TRCN13 MOVAS-1 cells were infected with lentiviruses expressing Acat (1 or ), Agpat (1,, 3,, or 5), Atf6, Dgat(1 or ), Gpat (1, 3 or ), Ire1, Lipin, Lpcat (1,, 3, or ), Lpgat1, Mboat1, Mogat, Scd or Sptlc1 shrna and selected with puromycin for 7 days. Each mrna level was measured by real-time qpcr. Changes in gene expression are reported as ratios relative to the control empty shrna.
3 Supplemental Table III. Fold changes of lipid contents in VSMCs treated with SCD inhibitor. Rank Fold change (vs. ) p value 1 PA-18:/18: PA-16:/16: PA-16:/18: 3.. PA-1:/18: LPA-18: LPA-16: DAG-16:/18: DAG-16:/16: PE-16:1/: PE-16:/: LPS-18: DAG-16:1/18: DAG-18:/18:.7. 1 DAG-1:/18: LPS-16: PE-16:/16: PE-16:/18: PS-17:/17: PS-18:1/: PS-16:1/18: LPA-16: PE-16:/: TAG-18:/18:/18: PE-18:3/: LPA-: PE-:/: PE-16:/18: PG-16:/: DAG-18:/18: PG-16:/16: PG-16:/: PE-16:1/18: PS-16:1/18: PE-18:/: TAG-16:/16:/18: PG-16:/16: PE-16:/: LPC-16:e PE-16:/18:.18.3 PE-16:1/16: PG-16:/:1..19
4 DAG-1:/18: LPA-18: PG-16:1/: PS-18:1/: LPS-: PE-18:/: PS-18:1/: LPS-16: PS-16:/18: LPC-18: PI-18:/18: DAG-16:/18: PA-18:/: PG-16:16: CER-18: DAG-16:/: PE-16:/18: PE-18:/18: LPA-: LPS-18: PE-16:1/: PS-18:/: PE-18:/: SM-: TAG-16:/16:/16: PA-16:/18: PC-36: PC-36: CE-: PE-16:1/: PE-18:1/18: LPA-18: PE-18:/: PE-18:/18: PE-16:1/18: PI-18:/: PS-:1/: PS-16:/18: PA-16:/16: CER-: PI-16:/16: PA-16:/: PS-18:1/: PE-16:1/18:
5 86 CE-: CER-: LPS-18: PE-16:/: PE-18:/: PE-18:1/: PE-18:/: DAG-16:1/18: DAG-18:/: PS-17:/17: PS-16:1/18: PE-16:/: PE-18:1/: PE-18:/: PE-18:/: PE-16:1/: PE-16:1/: PS-:/: LPI-18: PE-18:1/: PS-18:1/: PE-18:/18: PE-18:1/: PE-1:/18: TAG-16:/17:/18: LPI-18: SM-: PE-16:/16: PA-16:1/18: PC-36: PE-16:1/: PA-18:/; PE-18:3/: TAG-16:1/16:1/16: LPA-: PI-16:/: PE-16:/: PS-16:/16: PC-8: PE-18:1/18: SM-: DAG-18:1/: PE-18:/: DAG-18:1/18:
6 13 CER-16: DAG-16:/18: LPC-: PG-16:/18: LPC-18: SM-: PE-16:1/: PA-18:/18: SM-: PS-18:/: SM-: PA-16:1/18: PS-18:/: PE-18:1/: PG-16:/18: PE-18:1/: PE-18:/: PE-16:/: PA-18:/18: TAG-16:/16:1/17: CER-: PG-16:/18: PC-36: DAG-18:/18: PC-3: TAG-1:/17:/18: PE-:/: PG-18:/18: PG-16:/18: PE-18:1/18: PG-16:1/18: PC-3: PE-16:1/: PE-18:1/: PS-16:/18: SM-18: TAG-18:/18:/18: DAG-18:1/18: TAG-16:/18:/18: PE-16:/: PC-3: PC-3: PG-16:1/18: PG-16:/18:.73.5
7 17 PS-16:/18: PA-18:1/18: LPC-: TAG-16:1/16:1/18: PC-3: LPC-18:e PE-18:/18: LPC-16: CE-: TAG-1:/18:/18: PC-3: CE-18: PC-3: CE-16: TAG-18:1/18:1/: TAG-1:/16:/18: PC-3: PE-16:1/: PG-16:/18: PC-3: PC-8: PE-:/: PC-3: PC-38: TAG-1:/16:/18: PE-:1/:.6.16 TAG-16:/17:/18: PS-16:/18: PA-18:1/18: LPC-16:.6.5 PG-18:1/18: LPC-18: CE-18: PC-36: PC-8: PC-: PC-: PC-36: PC-36: PC-33: PC-3: LPC-18: PC-38: PC-:.5.
8 18 TAG-18:/18:/18: PC-38:7.5. TAG-15:/18:1/18: LPS-:3.9.7 TAG-15:/16:/18: LPC-: CE-18: PS-16:1/: LPC-:.7. 7 TAG-1:1/18:/18: TAG-1:/16:1/18: TAG-1:1/16:1/18: PC-3: PC-: DAG-18:1/: PC-38:6.. 3 PC-38: PC-36:.. 36 LPI-: PC-36: TAG-1:1/16:/18: TAG-18:1/18:1/:6.1.1 PC-38: CE-16:1..5 TAG-18:/18:1/18: PG-16:1/:1..3 LPI-: TAG-16:1/16:1/18: TAG-16:/16:/18: TAG-18:1/18:1/18: PG-18:1/18: PG-18:/18: LPI-: TAG-16:/16:1/18: TAG-16:/18:1/18: PS-18:/: TAG-18:1/18:/18: CE-: LPI-: PG-18:1/18: TAG-16:/18:/18: PE-18:/: LPC-: LPI-18:.6.19
9 6 TAG-16:/18:1/18: TAG-16:1/17:/18: TAG-16:1/18:1/18: TAG-16:1/18:1/18: LPC-: TAG-18:18:/18: TAG-18:1/18:1/18: LPI-18: PG-1:/18: PG-1:/18: TAG-17:/18:1/18: TAG-17:/18:1/18: TAG-1:1/18:1/18: TAG-16:1/18:1/18: TAG-16:/17:/18: PG-16:/: PG-16:1/: PG-18:/18:3..19 VSMCs were treated with 3nM CAY1566 for hours. Total lipids were extracted using Bligh & Dyer's method and analyzed with LC-MS/MS. CE; choletseryl ester, DAG; CER; ceramide, diacylglycerol, LPA; lysophosphatidic acid; LPC; lysophosphatidylcholine, LPE; lysophosphatidiyl ethanolamine, LPI; lysophosphatidylinsitol, LPS; lysophosphatidylserinepa; phosphatidic acid, PC; phosphatidylcholine, PE; phosphatidylethanolamine, PG; phosphatidylglycerol PI; phosphatidylinositol, PS; phosphatidylserine, SM; sphingomyelin; TAG; triacylglycerol,
10 Supplemental Table IV. Fold changes of lipid content in the medail layer of aortas from SMC-Scd1/ KO mice Rank Fold change (vs control) P value 1 PA-18:/18: 63.. PA-16:/18: PA-1:/18: PA-16:/16: PG-16:/16: PG-16:/: PE-16:1/: LPS-16: LPA-18: LPC-: DAG-16:/18: PE-16:1/: LPS-16: LPI-18: PI-16:/16: TAG-16:/17:/18: PG-16:1/18: PS-16:1/18: PE-:/:.5.3 DAG-16:1/18:.. 1 LPI-18: CE-: PS-16:/18:6.9.6 LPS-: PS-16:1/:..8 6 PS-18:/: DAG-18:/18: CER-18: PS-18:1/: PG-16:/18: DAG-1:/18: PS-18:1/: PS-16:/18:.. 3 PA-16:/: PG-18:/18: DAG-16:/16: DAG-18:/18: PS-16:/18: PE-18:/: LPI-18: LPI-: PG-16:16: PE-18:/: 1.8. PS-18:1/:
11 5 LPC-16:e SM-: PE-16:/: TAG-16:/16:/18: PE-16:/: LPE-18: LPA-16: TAG-18:/18:/18: PA-16:1/18: PG-16:1/: PS-16:/16: CER-: CE-18: LPC-18: LPC-16: PS-:/: PI-18:/18: LPI-: PE-16:/18: LPC-: PE-16:/: DAG-18:1/: LPE-18: PS-18:/: CE-16: PE-18:/18: SM-16: PS-18:1/: PE-16:1/: PE-16:1/18: PG-16:/18: LPA-: PC-36: PI-16:/: PE-16:/16: PE-18:/: PE-18:1/: PE-18:1/: PE-18:/: PE-18:/: SM-18: TAG-16:/16:/16: PA-18:/18: SM-: PG-16:/18: PI-18:/: LPC-: 1.1.5
12 9 PE-18:/18: PE-16:1/: PE-18:/18: PS-16:1/18: PC-36: DAG-18:/18: PE-16:1/: PE-16:/18: CE-: PE-16:/16: PE-18:/: LPC-18:e LPS-18: SM-: PE-16:/: PA-18:/18: SM-18: PE-18:/: CE-: SM-: PE-16:/18: PE-16:1/: PC-36: SM-: LPC-18: PE-16:/: PE-18:/: PC-8: PE-18:1/18: PA-16:/18: LPC-16: PE-18:1/: DAG-1:/18: TAG-1:/18:/18: LPC-18: PE-:/: LPA-18: PE-18:/: PI-18:/; PA-18:/: DAG-18:/: PS-16:/18: TAG-16:/18:/18: PE-18:/: PG-16:1/18: PS-16:/18: SM-:1.97.1
13 139 PC-36: PE-16:1/: PG-16:/: PS-18:1/: CE-18: DAG-16:/18: PS-18:/: PE-18:1/: PE-16:1/18: CE-: CER-: PE-:/: PA-18:/: SM-18: PE-18:3/: PE-16:/: TAG-16:/16:1/17: LPS-18: PS-:1/: PE-16:/18: PE-18:1/: PE-1:/18: PG-18:1/18: PS-16:1/18: PE-18:1/: LPC-: PG-18:1/18: CE-18: PE-18:/18: LPE-16: PE-16:1/: PS-16:/16: PS-18:/: PE-16:1/: LPC-: PC-3: PC-3: PE-18:3/: TAG-18:/18:/18: PE-18:1/18: PA-18:1/18: PE-16:1/18: PE-18:1/: CER-: DAG-16:1/18: PC-3: PC-3:.67.5
14 186 PE-18:1/18: PE-:1/: PC-3: TAG-18:/18:1/18: PC-8: PC-: DAG-18:1/18: CER-16: PC-3: PC-36: PC-3: DAG-16:/18: PC-3: PE-18:/: PC-38: TAG-16:1/16:1/18:.58.1 PC-3: DAG-18:1/18:1.57. PC-3: TAG-16:/17:/18: PC-33: PC-: TAG-1:/16:/18: LPA-18: PC-36: PC-8: PA-18:1/18: DAG-16:/: LPS-18: PC-3: PC-38: TAG-17:/18:1/18: TAG-17:/18:1/18: PC-36:6.8. LPS-: PE-16:/: PC-38: PC-36:5.6. TAG-1:1/18:/18: LPA-: LPC-18:..6 7 TAG-15:/16:/18: TAG-1:1/16:1/18:..1 9 LPA-16: TAG-1:/16:/18: PC-38: PC-38:5..
15 33 PG-16:1/: DAG-18:1/: PC-: TAG-16:1/17:/18: PG-18:/18: PE-16:1/16: TAG-16:/18:/18:..1 TAG-16:/16:/18: PC-38:.. PC-: CE-16:1.39. TAG-1:1/16:/18: PC-36: TAG-1:/17:/18: TAG-16:1/16:1/18: TAG-16:/17:/18: PA-16:1/18: PC-3: PG-1:/18: PG-18:1/18: TAG-16:1/18:1/18: TAG-16:1/18:1/18: LPC-: TAG-16:/18:1/18: TAG-16:/18:1/18: TAG-18:1/18:1/: TAG-16:/16:1/18: TAG-18:18:/18: TAG-18:1/18:1/18: TAG-1:/16:1/18: TAG-18:1/18:/18: TAG-16:1/18:1/18: TAG-18:1/18:1/: TAG-18:1/18:1/18: TAG-15:/18:1/18: TAG-16:1/16:1/16: TAG-1:1/18:1/18: TAG-18:/18:/18: PE-16:/: LPA-: CE-:1..7 Mice were sacrificed at 18-week-old. Total lipids in the aortic media were extracted by Bligh & Dyer's Method and were analyzed with LC-MS/MS. CE; choletseryl ester, DAG; CER; ceramide, diacylglycerol, LPA; lysophosphatidic acid; LPC; lysophosphatidylcholine, LPE; lysophosphatidiyl ethanolamine, LPI; lysophosphatidylinsitol, LPS; lysophosphatidylserinepa; phosphatidic acid, PC; phosphatidylcholine, PE; phosphatidylethanolamine, PG; phosphatidylglycerol
16 Supplemental Table V. qpcr primer list Gene Primer Bank ID Forward Reverse Acat1 639a1 GAAACCGGCTGTCAAAATCTGG TGTGACCATTTCTGTATGTGTCC Acat 157a1 ACAAGACAGACCTCTTCCCTC ATGGTTCGGAAATGTTGCACC Agpat1 6359a1 TAAGATGGCCTTCTACAACGGC CCATACAGGTATTTGACGTGGAG Agpat a1 CAGCCAGGTTCTACGCCAAG TGATGCTCATGTTATCCACGGT Agpat3 7978a1 CTGCTTGCCTACCTGAAGACC GATACGGCGGTATAGGTGCTT Agpat 796a1 CCAGTTTCTATGTCACCTGGTC GCAGAGTCTGGCATTGATCTTG Agpat5 7977a1 CACACGTACTCTATGCGCTAC AAGAAGAGCACCATGTTCTGG Alp a1 CCAACTCTTTTGTGCCAGAGA GGCTACATTGGTGTTGAGCTTTT Atf a1 GAGGATTTTGCTAACCTGACACC TTGACGGTAACTGACTCCAGC Atf a1 ATGGCGCTCTTCACGAAATC ACTGGTCGAAGGGGTCATCAA Awat1 1877c3 AGAGTGTGCCTAACACCACC CCGAACGTGAAGGTAGGGAC Awat 93a1 TTGACTGGAAGACCCCTGAG TCACCATCTTGAGTGGGAAGTA Chop a1 CTGGAAGCCTGGTATGAGGAT CAGGGTCAAGAGTAGTGAAGGT Dgat a1 TCCGTCCAGGGTGGTAGTG TGAACAAAGAATCTTGCAGACGA Dgat a1 GCGCTACTTCCGAGACTACTT GGGCCTTATGCCAGGAAACT Gadd a1 GAGGGACGCCCACAACTTC TTACCAGAGACAGGGGTAGGT Gpat a1 ACAGTTGGCACAATAGACGTTT CCTTCCATTTCAGTGTTGCAGA Gpat 63319a1 CACTGCTCCGAGGTTTTGATG AGGTTGGCAGCAATTCCATAC Gpat3 7376a1 GGCCTTCGGATTATCCCTGG CTTGGGGGCTCCTTTCTGAA Gpat 3531a1 AGCTTGATTGTCAACCTCCTG CCGTTGGTGTAGGGCTTGT GPAT 5151c GGTTAGGAGTGCTGATTCGGT CCAGAAGGCTAATCCCTGTGAA Lclat c1 TTTTATGCTCGGCCCCATTTT CACAAGACGGCTGCTAATCCA Lipin a1 CTCCGCTCCCGAGAGAAAG TCATGTGCAAATCCACGGACT Lipin a1 GAAGTGGCGGCTCTCTATTTC AGAGGGTTACATCAGGCAAGT LIPIN 769c1 TCTACAAGGGCATTAACCAGGC AACGTGAAAAGGTGAACACTGA Lipin a1 CAAACCTCGTGGTGAAAATCAAC CCACAGTGCTCTCAGGTAAGT Lpcat a1 GGCTCCTGTTCGCTGCTTT TTCACAGCTACACGGTGGAAG Lpcat 7375a1 CCCTTCGTCCAGCAGACTAC GCAGCAAAATTATTCCAACCAGT Lpcat a1 GACGGGGACATGGGAGAGA GTAAAACAGAGCCAACGGGTAG Lpcat c3 CTCATCCGATACCCCAACAGT GGGAGGAACTCTACATCCACG Mboat a1 AGCCTCTCTTACCGTACCACC GGCTGGCTTTACCAGGATGTA Mboat a1 TCAGACACGTAGTTGCTACCC TGCAGTAGGAAATCCCACTTTG Mboat7 3151a1 TACCGCACCTACCTGGATTG AGAAGACCGGGATCATGTAGAA Mogat a1 TGGTGCCAGTTTGGTTCCAG TGCTCTGAGGTCGGGTTCA Mogat 89798a1 TGGGAGCGCAGGTTACAGA CAGGTGGCATACAGGACAGA Ocn N/A GCAATAAGGTAGTGAACAGACTCC GTTTGTAGGCGGTCTTCAAGC Opn a1 AGCAAGAAACTCTTCCAAGCAA GTGAGATTCGTCAGATTCATCCG PiT a1 TTTGACAAACTTCCTCTGTGGG GGACTTTCAGACGGACTAGACTT SCD c1 TCTAGCTCCTATACCACCACCA TCGTCTCCAACTTATCTCCTCC Scd a1 TTCTTGCGATACACTCTGGTGC CGGGATTGAATGTTCTTGTCGT Scd a1 GCATTTGGGAGCCTTGTACG AGCCGTGCCTTGTATGTTCTG Scd a1 GTTGCCACTTTACTGAGATACGC GAAGCCCTCGCCCATACTT Scd 3179a1 GCCCACTTGCCACAAGAGAT GTAGCTGGGGTCATACAGATCA SCD c1 TGCGACGCCAAGGAAGAAAT CCTCCAGACGATGTTCTGCC Sm a1 CAACAAGGGTCCATCCTACGG ATCTGGGCGGCCTACATCA Smmhc 19578a1 GAGCAAACTCAGGAGAGGAAAC GTCCCGAGCGTCCATTTCTTC
17 Sptlc1 9577a1 ACGAGGCTCCAGCATACCAT TCAGAACGCTCCTGCAACTTG Sptlc a1 AACGGGGAAGTGAGGAACG CAGCATGGGTGTTTCTTCAAAAG sxbp-1 N/A GAGTCCGCAGCAGGTG GTGTCAGAGTCCATGGGA TetR N/A TGCTTAATGAGGTCGGAATCG ATCTCAATGGCTAAGGCGTC The primers for Ocn, sxbp-1 and TetR were designed in the laboratory.
18 Supplemental Table VI. 3-Qtrpa spectrometer settings for lipidomic analysis Lipid Name Q1 m/z Q3 m/z DP CE EP CXP CEP 1: CE 1: CE 15: CE 15: CE 16: CE 16: CE 17: CE 17: CE 18: CE 18: CE 18: CE 18: CE 18: CE 18: CE 18: CE : CE : CE : CE : CE : CE : CE : CE : CE : CE : CE : CE : CE : CE : CE : LPC 13: LPC 13: LPC 16:e LPC 16: LPC 16: LPC 16: LPC 18: LPC 18: LPC 18: LPC 18: LPC 18:e LPC : LPC o-: LPC : LPC o-:
19 LPC : LPC : LPC : LPC : LPC : LPC : LPC o-: LPC o-: LPC o-: LPS 13: LPS 16: LPS 16: LPS 18: LPS 18: LPS 18: LPS : LPS : LPS : LPS : LPG 13: LPG 16: LPG 16: LPG 18: LPG 18: LPG 18: LPG : LPG : LPG : LPG : LPI 13: LPI 16: LPI 16: LPI 18: LPI 18: LPI 18: LPI : LPI : LPI : LPI : LPI 13: LPI 16: LPI 16: LPI 18: LPI 18: LPI 18: LPI : LPI :
20 LPI : LPI : LPA 16: LPA 16: LPA 17: LPA 18: LPA 18: LPA 18: LPA : LPA : LPA : LPA : LPE 13: LPE 16:e LPE 16: LPE 16: LPE 18: LPE 18: LPE 18: LPE 18: LPE 18:e LPE : LPE : LPE : LPE : LPE : CER 1: CER 16: CER 18: CER : CER : CER : DHCer 16: DHCer 18: DHCer : DHCer : GlcCre 1: GlcCre 16: GlcCre 18: GlcCre : GlcCre : GlcCre : GlcCre : Cer1P 1: Cer1P 16: Cer1P : DHCer1P 1:
21 DHCer1P : SM 1: SM 16: SM 18: SM 18: SM 18: SM : SM : SM : SM : SM : SM : DHSM 18: DHSM : PC : PC 6: PC 8: PC 8: PC 8: PC 3: PC 3: PC 3: PC 3: PC 3: PC 3: PC 3: PC 33: PC 3: PC 3: PC 3: PC 3: PC 3: PC 3: PC 36: PC 36: PC 36: PC 36: PC 36: PC 36: PC 36: PC 36: PC 36: PC 38: PC 38: PC 38: PC 38: PC 38:
22 PC 38: PC : PC : PC : PC : PC o-3: PC o-3: PC o-3: PC o-3: PC o-3: PC o-3: PC o-3: PC o-3: PC o-3: PC o-36: PC o-36: PC o-36: PC o-36: PC o-36: PC o-36: PC o-38: PC o-38: PC o-38: PC o-: PC o-: PC 3p: PC 3p: PC 3p: PC 3p: PC 3p: PC 3p: PC 3p: PC 36p: PC 36p: PC 36p: PC 36p: PC 36p: PC 36p: PC 38p: PC 38p: PC 38p: PC p: PC p: PE 17:/17: PE 3: PE 3: PE 3:
23 PE 3: PE 3: PE 3: PE 3: PE 35: PE 35: PE 36: PE 36: PE 36: PE 36: PE 36: PE 38: PE 38: PE 38: PE 38: PE PE : PE : PE : PE : PE p: PE p: PE : PE : PE : PE : PE 3p: (16:/18:) PE 3p:1 (16:/18:1) PE 36p: (16:/:) PE 36p:3 (16:/:3) PE 36p: (18:/18:) PE 36p:1 (18:/18:1) PE 38p:6 (16:/:6) PE 38p:5 (16:/:5) PE 38p: (18:/:) PE p:6 (18:/:6) PE p:5 (18:/:5) PE p: (:/:) PE 3o: PE 3o: PE 36o: PE 36o: PE 36o: PE 36o: PE 36o: PE 36o: PE 38o:
24 PE 38o: PE o: PE o: PE o: PS 3: PS 3: PS 3: PS 3: PS 36: PS 36: PS 36: PS 38: PS 38: PS 38: PS : PS : PS : PS : PS : PI 3: PI 3: PI 3: PI 3: PI 3: PI 3: PI 36: PI 36: PI 36: PI 36: PI 36: PI 38: PI 38: PI 38: PI 38: PI 38: PI : PI : PI : PG 3: PG 3: PG 3: PG 3: PG 3: PG 36: PG 36: PG 36: PG 36:
25 PA 3: PA 3: PA 3: PA 3: PA 3: PA 3: PA 3: PA 36: PA 36: PA 36: PA 38: FFA 1: FFA 13: FFA 1: FFA 15: FFA 16: FFA 16: FFA 17: FFA 17: FFA 18: FFA 18: FFA 18: FFA 18: FFA 19: FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : MAG 1: MAG 1: MAG 15: MAG 16: MAG 16: MAG 17: MAG 17: MAG 18: MAG 18:
26 MAG 18: MAG 18: MAG 19: MAG : MAG : MAG : MAG : MAG : MAG : MAG : MAG : MAG : MAG : MAG : MAG : DAG 3: DAG 3: DAG 3: DAG 3: DAG 3: DAG 3: DAG 3: DAG 3: DAG 36: DAG 36: DAG 38: DAG 38: DAG 3: DAG 3: DAG 3: DAG 36: DAG 36: DAG 36: DAG 38: DAG : DAG 36: DAG 36: DAG 36: DAG 38: DAG 38: DAG : DAG 36: TAG 8: TAG 8: TAG 8: TAG 8: TAG 9:
27 TAG 5: TAG 5: TAG 8: TAG 8: TAG 5: TAG 5: TAG 5: IS TAG 9: TAG 51: TAG 8: TAG 5: TAG 5: TAG 5: TAG 9: TAG 5: TAG 51: TAG 51: TAG 51: TAG 5: TAG 5: TAG 5: TAG 5: TAG 8: TAG 5: TAG 5: TAG 51: TAG 5: TAG 5: TAG 53: TAG 51: IS TAG 5: TAG 5: TAG 5: TAG 5: TAG 5: TAG 5: TAG 56: TAG 58: TAG 5: TAG 5: TAG 56: MHC 16: MHC 18: MHC : MHC : MHC : MHC :
28 DHC 16: DHC 18: DHC : DHC : DHC : DHC : THC 17: THC 16: THC 18: THC : THC : THC : THC : GM3 16: GM3 18: GM3 : GM3 : GM3 : GM3 : LPS LPS-13: LPS LPS-16: LPS LPS-16: LPS LPS-18: LPS LPS-18: LPS LPS-18: LPS LPS-: LPS LPS-: LPS LPS-: LPS LPS-: LPI LPI-13: LPI LPI-16: LPI LPI-16: LPI LPI-18: LPI LPI-18: LPI LPI-18: LPI LPI-: LPI LPI-: LPI LPI-: LPI LPI-: LPA LPA-16: LPA LPA-16: LPA LPA-17: LPA LPA-18: LPA LPA-18: LPA LPA-18: LPA LPA-: LPA LPA-:
29 LPA LPA-: LPA LPA-: LPE LPE-13: LPE LPE-16:e LPE LPE-16: LPE LPE-16: LPE LPE-18: LPE LPE-18: LPE LPE-18: LPE LPE-18: LPE LPE-18:e LPE LPE-: LPE LPE-: LPE LPE-: LPE LPE-: LPE LPE-: LPG LPG-13: LPG LPG-16: LPG LPG-16: LPG LPG-18: LPG LPG-18: LPG LPG-18: LPG LPG-: LPG LPG-: LPG LPG-: LPG LPG-: PE 17:/17: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 3: PE 36: PE 36: PE 36: PE 36: PE 36: PE 36: PE 36:
30 PE 36: PE 36: PE 36: PE 36: PE 36: PE 36: PE 36: PE 36: PE 36: PE 36: PE 36: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE 38: PE PE PE PE PE PE : PE : PE : PE : PE : PE : PE : PE : PE : PE : PE : PE : PE :
31 PE : PE : PE : PE PE-3p: (16:/18:) PE PE-3p:1 (16:/18:1) PE PE-3p:1 (16:/18:) PE PE-36p: (16:/:) PE PE-36p:3 (16:/:3) PE PE-36p: (18:/18:) PE PE-36p:1 (18:/18:1) PE PE-36p:1 (18:/18:) PE PE-38p:6 (16:/:6) PE PE-38p:5 (16:/:5) PE PE-38p: (18:/:) PE PE-p:6 (18:/:6) PE PE-p:5 (18:/:5) PE PE-p: (:/:) PS PS-16:/16: PS PS-1:/18: PS PS-16:/16: PS PS-1:/18: PS PS-16:/18: PS PS-17:/17: PS PS-16:/18: PS PS-16:/18: PS PS-16:1/18: PS PS-16:1/18: PS PS-16:/18: PS PS-16:/18: PS PS-16:1/18: PS PS-16:/18: PS PS-17:/17: PS PS-16:/18: PS PS-16:/18: PS PS-16:1/18: PS PS-16:/: PS PS-16:/: PS PS-16:1/: PS PS-18:1/: PS PS-18:/: PS PS-16:/: PS PS-18:/: PS PS-18:/: PS PS-18:1/: PS PS-18:/: PS PS-18:1/: PS PS-18:/:
32 PS PS-18:1/: PS PS-18:/: PS PS-18:/: PS PS-18:1/; PS PS-:/: PS PS-;1/: PS PS-18:/: PS PS-18:3/: PS PS-:/: PS PS-18:/: PS PS-:/: PS PS-:1/: PI PI-16:/16: PI PI-18:/18: PI PI-16:/: PI PI-:/18: PI PI-18:/: PI PI-18:/: PI PI-16:/: PI PI-:/: PI PI-18:/: PI PI-18:/: PI PI-18:/: PG PG-1:/18: PG PG-16:/16: PG PG-1:/18: PG PG-16:/16: PG PG-16:/18: PG PG-17:/17: PG PG-16:/18: PG PG-16:1/18: PG PG-16:/18: PG PG-16:1/18: PG PG-16:/: PG PG-16:1/: PG PG-18:1/18: PG PG-18:/18: PG PG-16:/: PG PG-18:/18: PG PG-18:1/18: PG PG-16:1/: PG PG-18:/18: PG PG-18:1/18: PG PG-16:/: PG PG-16:1/: PA PA-1:/18: PA PA-1:/18:
33 PA PA-1:/18: PA PA-16:/16: PA PA-1:/18: PA PA-16:/16: PA PA-16:/18: PA PA-16:1/18: PA PA-16:1/18: PA PA-16:/18: PA PA-1:/: PA PA-16:/18: PA PA-17:/17: PA PA-16:/: PA PA-16:1/: PA PA-18:/18: PA PA-18:1/18: PA PA-16:/: PA PA-16:1/: PA PA-18:/18: PA PA-16:/: PA PA-18:/18: PA PA-18:/: PA PA-18:1/: PA PA-3: PA PA-3: PA PA-3: PA PA-3: PA PA-3: PA PA-3: PA PA-3: PA PA-36: PA PA-36: PA PA-36: PA PA-38: FFA 1: FFA 1: FFA 1: FFA 15: FFA 16: FFA 16: FFA 17: FFA 18: FFA 18: FFA 18: FFA 18: FFA 18: FFA : FFA :
34 FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA : FFA ; FFA : DP;Declustering potential CE; Collision energy EP; Entrance potential CXP; Collision cell exit potential CEP; Collision cell entrance potential
35 A B Klotho +/+ Klotho -/- C Klotho +/+ Klotho -/ Serum Phosphorus (mg/dl) FGF3 (pg/ml) Calcified lesion (µm ) Klotho +/+ Klotho -/- Supplemental Figure 1
36 Serum creatinine (µg/dl) Calcified lesion (%) Serum stearate (mg/ml) A B C D Sham CKD Serum phosphorus (mg/dl) Aortic Calcium (µg/mg) Scd mrna level (vs. sham) E F G H Scd1 Scd I J K Kidney function egfr Normal 8 ± 11.5 CKD 3 ± 6.5 Serum oleate (mg/ml) FGF3 (pg/ml) Normal 8 ± 11.5 CKD 3 ± 6.5 Desaturation index SCD activity (nmol/min/mg protein) 3 1 Sham CKD Normal 8 ± 11.5 CKD 3 ± 6.5
37 G Supplemental Figure 3 Scd mrna (relative mrna) Scd1 shrna Scd shrna p-eifα ATF CHOP GAPDH A E Scd1 mrna Scd mrna empty shrna Scd1 shrna empty shrna Scd shrna Tunica mycin Thapsig argin Calcium (mg/mg protein) Scd1 shrna Scd shrna H Relative Atf6 mrna level K Calcium (µg/mg) B C Empty shrnas Scd1 shrna empty shrna empty shrna Scd shrna empty shrna Atf6 shrna I Relative Ire1 mrna level L ALP activity (unit/mg protein) Scd1 shrna Scd shrna Relative mrna level Empty/Empty shrna Scd1/Scd shrna Scd1/Empty shrna Scd/Empty shrna Chop sxbp-1 empty shrna empty shrna empty shrna Atf6 shrna Ire1 shrna Perk shrna Calcium (µg/mg) Scd1/ shrnas F D empty shrna Ire1 shrna J Relative Perk mrna level M Calcium (µg/mg) empty shrna Perk shrna - +
38 % remaining mrna level Empty shrna Acat1 shrna Acat shrna Agpat1 shrna Agpat shrna Agpat3 shrna Agpat shrna Agpat5 shrna Dgat1 shrna Dgat shrna Gpat1 shrna Gpat3 shrna Gpat shrna Lpcat1 shrna Lpcat shrna Lpcat3 shrna Lpcat shrna Lpgat1 shrna Mboat1 shrna Mogat shrna Sptlc1 shrna Supplemental Figure
39 Ocn mrna level A B C GPAT1 GAPDH GPAT GAPDH D Calcium (mg/mg) Control shrna Control shrna Gpat1 shrna Gpqt shrna GPAT activity (nmol/min/mg) Control shrna Gpat shrna Gpat1 shrna Calcium (mg/mg) 18: F G H I J ATF CHOP GAPDH 6 Opn mrna level Gpat shrna E Empty shrna Gpat shrna K ATF CHOP GAPDH PiT1 mrna level EmptyshRNA EmptyshRNA 1 8 Gpat shrna Pi uptake pmol/mg protein/ min 6 L Atf mrna level M Chop mrna level N sxbp-1 mrna level Supplemental Figure 5
40 A B Dox GPAT GAPDH GPAT activity (nmol/min/mg) Dox +Dox C Calcium (µg/mg protein) D Alp mrna level G H I Atf mrna level Chop mrna level : ATF CHOP GAPDH Dox E PiT1 mrna level F Pi uptake pmol/mg protein/3 min Supplemental Figure 6
41 A 5 15 Agpat3 shrna G 5 Empty shrna ATF CHOP GAPDH 3 1 J Empty shrna 6 ATF CHOP GAPDH Supplemental Figure 7 Agpat5 shrna 8 Empty shrna Agpat5 shrna Agpat3 shrna 1 Empty shrna Agpat3 shrna I H PiT1 mrna level 1 F Ocn mrna level E Alp mrna level Ocn mrna level Alp mrna level 3 D Empty shrna PiT1 mrna level Calcium (µg/mg) 3 C B Empty shrna Agpat3 shrna Agpat5 shrna
42 A mrna expression (pg)/18s(ng) ND Lipin isoform 1 3 E Ocn mrna level H Atf mrna level B Chop mrna level F Opn mrna level Empty shrna K Lipin mrna level I C PAP activity (nmol/min/mg) PiT1 mrna level J sxbp-1 mrna level G Lipin shrna D Calcium (mg/mg) Empty shrna Lipin shrna 18: ATF CHOP GAPDH Supplemental Figure 8
43 A B 1 C-18: 1 C-18:1 NL 1.6 vehicle CER PA PE LPA PS PI PC SM LPC 1 C-18:1 incorporation (Fold change) PA LPA LPC PI NL PC CER PS PE SM C PA (µg/mg protein) sham 5/6nx 1:/18: 16:/16: 16:/18: 16:/: 16:/:1 16:/18: 16:/16:1 16:/18:1 16:1/18: 16:1/18:1 18:/18: 18:/18:1 18:/18: 18:/:3 18:/: 18:1/18:1 18:1/18: D PA content in ER (µg/mg ER protein) vehicle 1:/18: 16:/16: 16:/18: 16:/: 16:/:1 16:/18: 16:/16:1 16:/18:1 16:1/18: 16:1/18:1 18:/18: 18:/18:1 18:/18: 18:/:3 18:/: 18:1/18:1 18:1/18: Supplemental Figure 9
44 A Native Scd Chromosome Floxed Chromosome FLP-FRT Recombination Deleted Chromosome PGK- NEO B Scd1/ double lox icle i.p. x 5 Tamoxifen i.p. x 5 Control SMC-Scd1/ double conditional Smmhc-Cre ERT SMC-Scd1/ KO Supplemental Figure 1
45 Supplemental Figure 1. Serum phosphorous, serum FGF3 and vascular calcification in Klotho -/- mice. A) Phosphorous and B) FGF3 in SMMHC-GFP; Klotho -/- mice and SMMHC- GFP; Klotho +/+ mice (N=8). C) Quantification of calcified lesions in the aortic sinuses of SMMHC-GFP; Klotho -/- mice and SMMHC-GFP; Klotho +/+ mice. The lesions of aortic sinuses were cryosectioned and stained with von Kossa. Supplemental Figure. CKD reduces SCD activity in DBA/J mice and human patients. 8-week-old DBA/J male mice fed a chow diet (N=6) were 5/6 nephrectomized or shamoperated and fed a Western diet (TD11198, Harlan Teklad) for 1 weeks. A) Serum creatinine, B) serum phosphorus and C) FGF3 levels in sham-operated (blue) and 5/6 nephrectomized (red) DBA/J mice. D) Representative picture of aortas with von Kossa staining. E) Quantification of calcified lesions in the aortas of sham-operated and 5/6 nephrectomized DBA/J mice. The aortas were cryosectioned and stained with von Kossa. F) Aortic calcium content in sham-operated and 5/6 nephrectomized DBA/J mice. Aortic calcium level was analyzed using an ash assay coupled with a colorimetric assay. G) Levels of Scd1 and Scd mrna in the aortic medial layers of sham-operated and 5/6 nephrectomized DBA/J mice. H) Aortic SCD activity in sham-operated and 5/6 nephrectomized DBA/J mice. I) Serum stearate, J) serum oleate and K) desaturation index (18:1/18:) in age-matched human patients (N=1 per group) with normal kidney function and stage 3 and CKD was measured by gas chromatography using heptadecanoic acid as an internal standard. Other parameters were previously reported (9). p<.5 and p<.1 (-tailed Student s t-test). Supplemental Figure 3. Scd1 and Scd dual knockdown but not Scd1 and Scd single knockdown induces mineralization, osteogenic differentiation and ER stress of VSMCs. MOVAS-1 cells were treated with adenovirus containing Scd1 shrna and lentivirus containing Scd shrna. For the dual knockdown, Scd stable knockdown MOVAS-1 cells were treated with adenovirus containing Scd1 shrna at a multiplicity of infection (MOI) of. A) Scd1 and Scd mrna levels in Scd1 and Scd dual knockdown cells. Control cells and Scd stable knockdown cells were treated with adenovirus containing SCD1 shrna or empty shrna for hours. Scd1 and Scd mrna levels were analyzed with real-time qpcr. B) Calcium deposit. Cells treated with empty shrnas and Scd1/ shrna were stained with Alizarin red. C) Calcium content in Scd1 and Scd dual knockdown cells. Control cells and SCD stable knockdown cells were treated with adenovirus containing Scd1 shrna or empty shrna for 7 days in the 1
46 presence of. mm inorganic phosphate. Calcium was extracted with.1n HCl and analyzed with a colorimetric assay. D) ALP activity in Scd1 and Scd dual knockdown cells. Scd stable knockdown cells were treated with adenovirus containing Scd1 shrna or empty shrna for days. E) Immunoblot analysis of ER stress markers. Control cells and Scd stable knockdown cells were treated with adenovirus containing Scd1 shrna or empty shrna for hours. F) mrna levels of ER stress markers. Control cells and Scd stable knockdown cells were treated with adenovirus containing Scd1 shrna or empty shrna for hours. G) ER stress inducers tunicamycin and thapsigargin induce mineralization of VSMCs. VSMCs were treated with.5 g/ml tunicamycin and.5 M thapsigargin for 7 days. Calcium was analyzed with a colorimetric assay. H-J) Atf6, Ire1 and Perk shrnas efficiently reduced their target genes. K-M) Atf6-, Ire1- and Perk-knockdown attenuated SCD inhibition-induced mineralization of VSMCs. Atf6, Ire1 and Perk mrna levels were analyzed with real-time qpcr. Cells were treated with vehicle () or SCD inhibitor () for 7 days in the. mm inorganic phosphate. p<.5 and p<.1 vs. MOVAS cells treated with lentivirus containing empty shrna and adenovirus containing empty shrna or vehicle () ( tailed Student s t-test). Supplemental Figure. Acyltransferase-knockdown stable VSMCs. ShRNA-mediated knockdown of acyltransferases in VSMCs. MOVAS-1 cells were infected with lentiviruses containing acyltransferase shrnas for 8 hours and selected with 5 g/ml puromycin to generate each acyltransferase stable knockdown VSMC. Levels of acyltransferase mrnas were determined by real-time qpcr. Each shrna reduced its targeted acyltransferase by over 75%. N=3. Supplemental Figure 5. Gpat but not Gpat1 knockdown inhibits SFA inducedmineralization, osteogenic differentiation and ER stress in VSMCs. A) Immunoblot analysis of GPAT1 and GPAT in Gpat knockdown cells. MOVAS-1 cells were treated with Gpat1 and Gpat shrna. A single colony was isolated from MOVAS-1 cells infected with each lentivirus in the presence of puromycin. B) GPAT activity in Gpat knockdown cells. Total GPAT activity was measured in total cell lysates of Gpat1 and Gpat knockdown MOVAS-1 cells. C) Stearateinduced mineralization in Gpat1 and Gpat knockdown cells. Gpat1 and Gpat knockdown cells were treated with M stearate (18:) for 7 days. D and E) -induced mineralization in Gpat1 and Gpat knockdown cells. Gpat1 and Gpat knockdown cells were treated with either vehicle or 3 nm for 7 days. Cells were stained with Alizarin red. F) Ocn, G) Opn and H)
47 PiT1 mrna levels in Gpat1 and Gpat knockdown cells treated with. Gpat1 and Gpat knockdown cells were treated with 3 nm for hours. I) Phosphate uptake of Gpat knockdown cells treated with for 16 hours. J) Immunoblot analysis of stearate-induced ER stress markers in Gpat knockdown cells. Gpat knockdown cells were treated with for hours. K) Immunoblot analysis of -induced ER stress markers in Gpat knockdown cells. Gpat knockdown cells were treated with 3 nm for hours. L-N) mrna levels of ER stress markers such as Atf, Chop and sxbp-1 in Gpat1 and Gpat knockdown cells treated with. Gpat1 and Gpat knockdown cells were treated with 3 nm for hours. White colmun; empty shrna, Red column; Gpat shrna, Black column; Gpat1 shrna p<.1 (one-way ANOVA). Supplemental Figure 6. Human GPAT overexpression augments SFA inducedmineralization, osteogenic differentiation and ER stress in VSMCs. A) Immunoblot analysis of GPAT. MOVAS-1 cells overexpressing human GPAT were generated using a Tet-ON system. MOVAS-1 cells were treated with lentivirus containing tetr and human GPAT. A single colony was isolated from MOVAS-1 cells infected with lentivirus in the presence of hygromycin and zeocin. MOVAS-1 cells containing GPAT were treated with either vehicle or 1 mg/ml doxycycline (Dox) for hours. B) GPAT activity. MOVAS-1 cells containing human Gpat were treated with either vehicle or 1 mg/ml Dox for hours. Total GPAT activity was measured in the total cell lysates of GPAT overexpressed MOVAS-1 cells. C) Stearate and -induced mineralization in GPAT overexpressed cells. MOVAS-1 cells containing GPAT were treated with either vehicle or 1 mg/ml Dox for hours and then treated with M 18: and 3nM for 7 days. D) Alp and E) PiT1 mrna levels in Gpat overexpressed cells. F) Phosphate uptake in GPAT overexpressed cells. MOVAS-1 cells containing Gpat were treated with 1 mg/ml Dox for hours and then treated with 3nM for hours. G) Atf and H) Chop mrna levels in GPAT overexpressed cells. I) Immunoblot analysis of ER stress markers. MOVAS-1 cells containing GPAT were treated with 1 mg/ml Dox for hours and then treated with 3nM for hours. White column; without Dox treatment, Black column; with Dox treatment, p<.1 (one-way ANOVA). Supplemental Figure 7. Agpat3 and Agpat5 knockdowns inhibit SFA-induced mineralization, osteogenic differentiation and ER stress in VSMCs. A) SCD inhibition induced-mineralization in Agpat3 and Agpat5 knockdown cells. Agpat3 and Agpat5 knockdown 3
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