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SUPPLEMENTAL DATA

Manuscript title: Naturally occurring variants of human aldo-keto reductases with reduced in vitro metabolism of daunorubicin and doxorubicin Authors: Onkar S. Bains, Thomas A. Grigliatti, Ronald E. Reid, and K. Wayne Riggs Journal name: Journal of Pharmacology and Experimental Therapeutics

TABLES

Supplemental Table 1—Primers for creating non-synonymous single nucleotide polymorphic variants of human AKRs and CBR4 using site-directed mutagenesis. The base pair mutation is given beneath each variant [in square brackets]. Also, the mutated base pair is underlined in the forward and reverse primer sequences.

ENZYME VARIANT FORWARD PRIMER (5'3') REVERSE PRIMER (5'3') I15F CAAGATGCCCTTCCTGGGGTT CCAACCCCAGGAAGGGCATC AKR1B1 [AT] GG TTG H42L CGGGTACCGCCTCATCGACTG GGGCACAGTCGATGAGGCGG [AT] TGCCC TACCCG L73V GCGTGAGGAGGTCTTCATCGT GCTGACGATGAAGACCTCCTC [CG] CAGC ACGC K90E GGGCCTGGTGGAAGGAGCCTG GGCAGGCTCCTTCCACCAGGC [AG] CC CC G204S GCCAGTCCAAAAGCATCGTGG GTCACCACGATGCTTTTGGAC [GA] TGAC TGGC T288I CCAGGATATGACCATCTTACTC GTAGCTGAGTAAGATGGTCAT

[CT] AGCTAC ATCCTGG P87S CACTTTCTTTGAGAGATCCCTT CTTTCCTCACAAGGGATCTCT AKR1B10 [CT] GTGAGGAAAG CAAAGAAAGTG M286T GTGATGAGGAGACGGCAACCA GAGTATGGTTGCCGTCTCCTC [TC] TACTC ATCAC

N313D GACTATCCCTTCGATGCAGAA CAATATTCTGCATCGAAGGGA

[AG] TATTG TAGTC R170H CCAACTTCAACCACAGGCAGC CTCCAGCTGCCTGTGGTTGAA AKR1C1 [GA] TGGAG GTTGG Q172L CTTCAACCGCAGGCTGCTGGA GGATCATCTCCAGCAGCCTGC [AT] GATGATCC GGTTGAAG F46Y CAATAGAAGCCGGGTACCACC GAATCAATATGGTGGTACCCG AKR1C2 [TA] ATATTTGATTC GCTTCTATTG JPET#173179 2

H5Q GGAAGAATGGATTCCAAACAG CATTTAGCTTTACACACTGCT AKR1C3 [CG] CAGTGTGTAAAGCTAAATG GTTTGGAATCCATTCTTCC R66Q GGTTGGACTGGCCATCCAAAG CATCTGCAATCTTGCTTTGGA [GA] CAAGATTGCAGATG TGGCCAGTCCAACC A106T GGAAAACTCACTGAAAAAAAC GTCAACATAGTCCAATTGAGT [GA] TCAATTGGACTATGTTGAC TTTTTTCAGTGAGTTTTCC R170C GGGTGTCAAACTTCAACTGCA CATCTCCAGCTGCCTGCAGTT [CT] GGCAGCTGGAGATG GAAGTTTGACACCC P180S GAGATGATCCTCAACAAGTCA GCTTGTACTTAAGTCCTGACT [CT] GGACTTAAGTACAAGC TGTTGAGGATCATCTC GCCACTACCAAAAGATGAAAA CCACTGTGTCGAATATTACTT G135E AKR1C4 TGAAAAAGTAATATTCGACAC TTTCATTTTCATCTTTTGGTAG [GA] AGTGG TGGC S145C CAGTGGATCTCTGTGCCACAT CTCCCATGTGGCACAGAGATC [CG] GGGAG CACTG L311V GATATGTTGTCATGGATTTTGT GATAATCAGGATGGTCCATAA [CG] TATGGACCATCCTGATTATC CAAAATCCATGACAACATATC V135M CTGCAGTGTCCCCAAATGGAC GGTAGAAGAGGTCCATTTGG AKR7A2 [GA] CTCTTCTACC GGACACTGCAG Q157H GCATGCCTGCCACCGGCTGCA CTGGTGCAGCCGGTGGCAGG [GC] CCAG CATGC E180K GCTGGGAAGTGGCCAAGATCT GAGGGTACAGATCTTGGCCAC [GA] GTACCCTC TTCCCAGC G198S CCACTGTGTACCAGAGCATGT GGCGTTGTACATGCTCTGGTA [GA] ACAACGCC CACAGTGG S255N CGCTTCTTTGGGAATAACTGG GTAGGTCTCAGCCCAGTTATT [GA] GCTGAGACCTAC CCCAAAGAAGCG L70M CATTTGAAGAGATGGAGAAAC CTCGACCTAAATGTTTCTCCA CBR4 [CA] ATTTAGGTCGAG TCTCTTCAAATG

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FIGURES

Supplemental Fig. 1—Purification of human recombinant 6x-His tagged (A) AKR1B1, (B) AKR1B10, (C) AKR1C1, (D) AKR1C2, (E) AKR1C3, (F) AKR1C4, (G) AKR7A2, and (H) CBR4 wild-type enzymes. (Left) Gels stained with SYPRO® Ruby following SDS-PAGE showing purified protein samples (lane 3; 3 µg), free of contaminating proteins from bacterial lysates (lane 1; 10 µg total protein). Removal of contaminating proteins is observed in fractions from Qiagen purification procedures [Ni-NTA column flow through (lane 2; 10 µg total protein)]. (Right) Western blot detection of transformed lysates (lane 6) and purified protein samples (lane 8), confirms expression of the desired AKR protein with mobility at expected molecular weight (41.9 kDa for AKR1C3, 39.7 kDa for AKR1C4 and 39.3 kDa for AKR7A2). Little immunoreactivity was detected in the flow through samples (lane 7) suggesting that majority of the enzymes were bound to the Ni-NTA resin prior to their elution. GST-tagged AKR recombinant proteins (Abnova Corporation) were used as a positive controls for antibody immunoreactivity (lane 5; 1 μg). No antibody immunoreactivity is observed for untransformed bacterial lysate (lane 4; 10 μg total protein). M refers to the molecular weight markers.

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Supplemental Fig. 2—Representative Western blot detection of purified 6x-His tagged AKR proteins [(A) AKR1C3, (B) AKR1C4, and (C) AKR7A2 wild-type enzymes; lane 2; 3 µg] and native AKR proteins (lane 3; 3 µg). The 6x-His tagged proteins were incubated with Factor Xa for 6 hrs at 23oC to allow for removal of the tag and linker from the amino terminus. GST- tagged AKR recombinant proteins (Abnova Corporation) were used as a positive controls for antibody immunoreactivity (lane 1; 1 μg). M refers to the molecular weight markers.

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Supplemental Fig. 3—Representative Western blot detection of cytosolic AKR and CBR proteins in pooled human liver (L) and human heart (H) lysates (20 μg total protein). Lysates were run on 18% SDS-PAGE gels and subjected to Western blotting for detection of AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR7A2, CBR1, and CBR3 proteins (ranging from 30 to 38 kDa). In addition to the reductase enzymes, β-tubulin band was also detected using Western blotting (~55 kDa). The 6x-His tagged purified proteins were used as positive controls (PCs) for antibody immunoreactivity for these enzymes. M refers to the molecular weight markers.