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Supplementary File 1: Target areas of each of the CYP2D siRNAs. Homologous areas of each of the CYP2D isoforms to each of the siRNAs were determined by CLUSTALW. CYP2D-KD1 and CYP2D-KD2 were purposely designed to target multiple CYP2D isoforms. Cyp2d10-Designs A and B were targeted to only Cyp2d10. Cyp Cluster sequences of Musmusculus Cyp2dcDNA Family Cyp2d9 TCATAGGGCAGGTGAGGCA CCACATGCCC 1146 TCCAGAGATGGCAGACCAGGC Cyp2d12 TCATAGGGCAGGTGAGGCA TCCAGAGATGGCAGACCAGGC CCACATGCCC 1120 Cyp2d10 TCATAGGGCAGGTGCGGCA TCCAGAGATGGCAGACCAGGC TCGTATGCCC 1141 Cyp2d11 TCATAGGGCAAGTGCAGCA TCCAGAGATGGCAGACCAGGC TCGCATGCCC 1146 Cyp2d - KD1 Cyp2d34 TCATAGGGCAGGTGCGGTG TCCAGAGATGGCAGACCAGGC CCGCATGCCC 1121 Cyp2d26 TCATAGGGCACGTGCGGCA TCCAGAGATGGCAGACCAGGC CCGCATGCCC 1143 Cyp2d22 TCATAGGGCAGGTGCAGTG TCCAGAGATGGCAGACCAGGC TCGCAT GCCC 1110 Cyp2d13 TCATAGGGCAGGTGCGGTG TCCAGAGATGGCAGACCAGGC CCACATGCCC 1143 Cyp2d40 TCATAGGGCAGGCACGGCG TCCAGAGATGGCAGACCAGGC CCGCATGCCC 597 Cyp2d9 CCTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCTTCTCAGTGCCTGATG 1495 Cyp2d12 CCTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCTTCTCAGTGCCTGATG 1469 Cyp2d10 CCTCTTCTTCAC G TGCCTCCT GCAGCACTTTAGCTTCTCAGTGCCCAATG 1490 Cyp2d11 CCTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCATCTCAGTGCCTGATG 1495 Cyp2d - KD2 Cyp2d34 CCTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCTTCTCAGTGCCAGCTG 1470 Cyp2d26 CCTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCTTCTCAGTGCCCGATG 1492 Cyp2d22 CCTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCATCTCAGTGCCCGATG 1459 Cyp2d13 CCTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCTTCTTAGTGCCTGCTG 1493 Cyp2d40 C CTCTTCTTCACCTGCCTCCT GCAGCGCTTTAGCTTCTCAGTACCGGATG 946 Cyp2d9 TCCTGAGGTTCTTAAT GCA TT CCCGATACTCTTGCGTATCCCAAGGCTGGCTG 796 Cyp2d12 TCCTGAGGTT A TTAA C A CA TT CCCGATACTCCTGCACATCCCAAGGCTGGCTG 770 Cyp2d10 TCCTGAGGTTCTTAATATGTT CCCCATACTCCTGCGCATCCCAGGACTGCCTG 791 Cyp2d11 TCCTGGGGT A CTTAAT GAG TT CCCAATATTCCTGCGCATCCCAGGGCTGGCTG 796 Cyp2d10 - Cyp2d34 TCCTG G GGTTCTTAA C A C GTT CCCAATACTCCTGCGAATCCCAGGGCTGGCTG 771 Design A Cyp2d26 CGGA G A GGT G CT G AAT GCCA T CCCAATGCTACTACACATCCCTGGTTTGCCTG 793 Cyp2d22 A CC CA T G TTC CT G AAT G TGTT CCCGATGCTCCTGCGCATCCCGGGGCTGGTTG 760 Cyp2d13 TCC A GAGGTTCT G AA C A CA TT TCCAATTCTCCTGCACATCCCAGGGTTGGCTG 793 Cyp2d40 T TACAA GGTT G T G AA G ATGTT CCCAATTGTCCTGCGCATCCCAGGGTTGGCTG 247 Cyp2d9 ACAAGGCCCT CCAAGGTCAGAAGTCCTT CA T CGCCATACTGGATAACCTG 846 Cyp2d12 ACAAGTTCCTG CAA A GTCAGAAGTCCTT CA T CGCCATAGTGGATAACCTG 820 Cyp2d10 GGAAGGTCTT CCAAGGTCAGAAGTCCTTACT GGCCATAGTGGAGAATCTG 841 Cyp2d11 ACATGGTCTT CCAAGGTCAGAAGTCCTT CA T GGCCATACTGGATAACCTG 846 Cyp2d10 - Cyp2d34 ACATGGTCTT CCAA A GTCAGAAG A CCT T CA T GGCCATACTGGATAACCTA 821 Design B Cyp2d26 ATAAAGCCTT CC CC A AGCT GAA T TCATT CA T AGCCTTAGTGAATAAGATG 843 Cyp2d22 GCAAGGTCTT CC CTG G GA A A A G G G CCTT TG T TACCATGTTGGATGAGCTG 810 Cyp2d13 ACAAAGTCTT CC CTG G G CA A AAGAC A TT T CT CACCCTGGTAAATAAGCTG 843 Cyp2d40 ATAAAATCTT CC CTG G G CA A AAG A CATT T CT CACCATGGTGGATAAGCTG 297 Accession numbers are as follows: Cyp2d9 NM_010006; Cyp2d10 NM_010005; Cyp2d11 NM_001104531; Cyp2d12 NM_201360; Cyp2d13 NM_003552; Cyp2d22 NM_019823; Cyp2d26 NM_029562; Cyp2d34 NM_145474; Cyp2d40 NM_023623.
Suppl File 2: DNA sequences of synthetically prepared siRNA constructs. *Predicted efficiency: Percentage of mRNA remaining in cells after siRNA directed cleavage
SupplFile 3: CYP2D primers used for PCR and qPCR. Primer sequences were obtained from other sources or designed using the Primer3 program available at http://wwwgenome. wi.mit.edu/cgi-bin/primer/primer3_www.cgi. a Indicates a PCR primer and not used for qPCR
HEPA1C WT-M RIIIPrI WT-M A Cyp2d HEPAIC RΙΙΙPrΙ WT-M B Cyp2d10 Cyp2d11 Cyp2d22 Cyp2d26 WT-M Hep C Hep WT-F WT-M D Cyp2d9 Hep WT-M Cyp2d10 Cyp2d11 Cyp2d22 Cyp2d26 Suppl File 4: CYP2D expression in murine cell lines and mouse primary hepatocytes. (A) Protein expression of CYP2Ds in the mouse hepatoma cell lines, HEPA1C1C7 and RIIIPrI as determined by Western blotting. Microsomes from wild-type male mouse livers (WT-M) were used as positive controls. (B) PCR detection of individual Cyp2ds in the HEPA1C1C7 and RΙΙΙPrΙ cell lines. (C) Western blot detection of Cyp2d protein in mouse primary hepatocyte S9 fraction compared to microsomes from WT-M mouse livers. (D) PCR detected Cyp2d10, Cyp2d11, Cy2d22 and Cy2d26, but not Cyp2d9 in male primary hepatocytes. Hep = primary hepatocytes; WT = wild-type; M = male; F = female.
1.2 * 0.8 * Relative Expression 0.4 0.0 SC 2 A 2 SC 6 A 6 SiRNA-A Supplementary File 5a Cyp2d10 Supplementary File 5: Repression of CYP2D isoforms in primary mouse hepatocytes by 2 or 6mM siRNAfollowing transfection with SureFecttransfection reagent. Cells were either transfected with the scrambled construct (SC), or transfected with the Cyp2d10-siRNA-A, Cyp2d-siRNA-B, Cyp2d-KD1 (siRNA-KD1), or Cyp2d-KD2 (siRNA-KD2) constructs. Expression of Cyp2d10 (1a), Cyp2d11 (1b), Cyp2d22 (1c), or Cyp2d26 (1d) was measured by qPCR.
Supplementary File 5b Cyp2d11
Supplementary File 5c Cyp2d22
Supplementary File 5d Cyp2d26
SureFECT TransIT- siQUEST TransIT- TKO Suppl File. 6: Comparison of the transfection efficiency of three different transfection reagents in primary mouse hepatocytes; TransIT-TKO and TransIT-siQUEST from Mirus Bio and SureFECT from SABiosciences were used to transfect cells with fluorescence labeled siRNA probes and visualized using confocal microscopy (40X objective). TransIT-siQUESThad the best transfection efficiency (nearly 100%), but this reagent also caused significant toxicity and repression of CYP2D isoforms was not observed. TransIT-TKO had reasonable transfection efficiency with little toxicity, while SureFECT appeared to work well, but the relatively high efficiency led to high toxicity. Both had transfection efficiencies approaching 90%.
