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Degenhardt et al: PPAR controls heme biosynthesis-Supplement
SUPPLEMENTARY MATERIAL
TABLES
Tab. S1: Information about the donors of the primary human hepatocytes. Surgical liver
biopsies were obtained from six individual donors including both male and female, who went
under surgery due to metastasis after informed consent was obtained. Hepatocytes were
isolated with the two-step collagenase perfusion method.
Donor
Sex
Age
Diagnosis
Donor1
Male
63
Colo-rectal metastasis
Donor 2
Male
44
Metastasis
Donor 3
Female
70
Hepatic metachrone lesion
Donor 4
Female
54
Metastasis
Donor 5
Male
73
Hepatocellular carcinoma
Donor 6
Male
73
Endocrine carcinoma
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Degenhardt et al: PPAR controls heme biosynthesis-Supplement
Tab. S2: Real-time PCR primers. The sequence, annealing temperature and the resulting
product size of the primer pairs for all genes used in this study are indicated.
Primer pairs (5’-3’)
Gene
Annealing
temperature
Product size
(bp)
RPLP0
AGATGCAGCAGATCCGCAT
GTGGTGATACCTAAAGCCTG
57-66 °C
318
ALAS1
AGGCCAAGGTCCAACAGACT
66 °C
217
ACCTCTTTCCTCACGGCATTC
ALAD
TGATGACATACAGCCTATCACCA
GGGACGCCAAAGATCAAGACA
60 °C
123
HMBS
TGTTTACCAAGGAGCTTGAACAT
GGCAGGGTTTCTAGGGTCTTC
64 °C
187
UROS
CTCAAGGACAAAGGGATTGCC
CCCCTGCTGGGAATAGTAGC
64 °C
102
UROD
AAGAGCAGGACCTAGAACGC
CGTCCAGCCAGTCGTTGTC
60 °C
103
CPOX
TGTTATCCACCCCAAGAATCCT
64 °C
131
TCAAGTATGTTGGAGTGAGGTCA
PPOX
CCCCCTTCTCCAAACCTCTG
GCCCAGTAATATGGAACGATGG
66 °C
242
FECH
GAGAGAGATGGCCTAGAAAGGG
CCACAGACATCGGCAGTGA
57 °C
277
Tab. S3: siRNA sequences. The sequence of the three siRNA oligonucleotides and their
position in the PPAR mRNA are indicated.
Name
Sequences (5’-3’)
Position
PPAR siRNA1
GCUUUGGCUUUACGGAAUA
323
PPAR siRNA2
CUUGGACCUGAACGAUCAA
1077
PPAR siRNA3
CCACCCGGACGAUAUCUUU
1425
Control siRNA
UGCGCUACGAUCGACGAUG
3
Degenhardt et al: PPAR controls heme biosynthesis-Supplement
Tab. S4: Genomic PCR primers. Sequences and locations of the primer pairs used for PCR
amplification of genomic regions containing the genes TSS and the ALAS1 REs. Positions are
indicated relative to the respective annotated gene TSS.
Gene-region
Location
(relative to
TSS)
-95 to +114
ALAS1-TSS
human
ALAD-TSS
-178 to +85
human
HMBS-TSS
-95 to +198
human
UROS-TSS
-104 to +105
human
UROD-TSS
-192 to +187
human
CPOX-TSS
-81 to +339
human
PPOX-TSS
-96 to +105
human
FECH-TSS
-255 to +170
human
ALAS1-RE1
-9111 to -8865
human
ALAS1-RE2
-2587 to -2061
human
ALAS1-RE3
-1073 to -670
human
ALAS1-RE4/5 +214 to +840
human
ALAS1-RE6 +4233 to +4621
human
ALAS1-RE7 +9687 to +9812
human
Alas1-TSS
-185 - +194
mouse
Alad-TSS
-99 - +280
mouse
Hmbs-TSS
-192 - +100
mouse
Uros-TSS
-112 - +185
mouse
Urod-TSS
-111 - +198
mouse
Cpox-TSS
-173 - +97
mouse
Ppox-TSS
-180 - +198
mouse
Fech-TSS
-184 - +93
mouse
Annealing
temperature
Sequences (5’-3’)
58 °C
CTTCTTCCTCCATGTCCAAG
GAAGTCCAAACGAAACGCTC
GCACAGCACAATCTGGGGAC
AGAAGACCGCCTTCAGGGG
TGAACAGAGCAAAGGAAGCG
GAAAGTAGGCTGTGTGTGGG
GCAGGAGCAATGACAACAGG
TCCCCTTCCCGTAGTGGTTC
GGATACCCAGACTGTCAGATG
GATAGAGTCCTGAGTTGGAAG
GTTCCCGCACTATCACCTGG
GATCTCTGCCACATTTCCGC
CCGCCTCTGCCAGTTCAATG
AGGTGCTCTGCTTGCCACAC
CCACGCCTTCTCAGGGATC
AGGAGTCCAGCAGGTTTTGC
GCTTACATCCTGACTCCAGC
GCTTATCAGCATCCACGATC
CCCACCCAGACTCATCACTG
ATCCCAACCACGAGGAAGGC
CCAGGTGCCTCCTCTGAAG
ATCCAGGGTTGTCTGAAGCC
GTGTCCTGCCAGCCGTGG
ATCAAGGGGGCGGAAGGAT
CCTGGGGTACAGAGCAAGAC
CTCACGGCATTCATTTCCTG
GCTCCATCCGTATTGCTGTA
GCCTAGTAAAAGCCAGAATAG
TGAGGTAGCCCGAGAGGTAG
TAGTCCCAGGAGTTGGAGAG
GCATAGGGACAGCGTCTCC
GGGCTCTCGGGCAACTCTG
GTGCGGGTCATCGGTCACC
GAGCGAAGAGGCTGGCTATG
CCAAACCACTACACACAGAG
ATTCCATTTCCCACCGCTCG
TCAGCGACTTGGGTTCAGAC
GTTTGGGTCCTACGGGCAGC
CACTTTTGCTCACTGTGGGTG
GCTGCTGCGAGTGTAGGTC
AAGTGAGTGACTGATAGAGGC
GCTCCACGGAACTGACTCC
GAACGCAGACGCAACCCAAC
CCCCAGCCTACAAGCCCCG
62 °C
62 °C
62 °C
58 °C
60 °C
57 °C
62 °C
62 °C
62 °C
60 °C
64 °C
66 °C
62 °C
64 °C
64 °C
64 °C
58 °C
64 °C
65 °C
64 °C
64 °C
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Degenhardt et al: PPAR controls heme biosynthesis-Supplement
Tab. S5: Sequences of putative PPREs in the ALAS1 gene. The PPRE name, its core
sequence (hexameric binding motifs in bold), the location relative to the TSS and the
conservation between human and mouse are indicated.
Name
Sequences (5’-3’)
Location
Conservation
RE1
TGAGTGGAAAGGTCA
-8976
not conserved
RE2
CTGGGTCAAAATCAA
-2369
77 %
RE3
CCATGCCAAAGGTCA
-737
not conserved
RE4
GTGGGGGATGGGGTC
+253
85 %
RE5
ATGAGGGAAAGGTCA
+818
not conserved
RE6
TAAGGCCAAGGTCCA
+4421
77 %
RE7
TTGGGTGAAAGTGGA
+9783
not conserved
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Degenhardt et al: PPAR controls heme biosynthesis-Supplement
FIGURE LEGENDS
Fig. S1: PPAR ligand control and basal expression. HepG2 cells were stimulated with 50
M of the PPAR ligand Wy14,643. RNA was extracted and quantitative real-time PCR was
performed for the eight heme biosynthesis genes (A). Columns indicate the means of at least
three independent treatments and the bars represent standard deviations. Two-tailed, paired
Student’s t-tests were performed to determine the significance of the mRNA induction by
PPAR agonist relative to solvent controls (* p < 0.05, ** p < 0.01, *** p < 0.001). Basal
mRNA expression levels of the genes relative to RPLP0 were measured by quantitative realtime PCR (B) in HepG2 cells.
Fig. S2: siRNA controls. HepG2 cells were transfected with either control siRNA or siRNA
specific for PPARand incubated for 48 h (A). RNA was extracted and quantitative real-time
PCR for the three PPAR subtypes. Two-tailed, paired Student’s t-tests were performed to
determine the significance of PPAR siRNA knock-down relative to control siRNA (* p <
0.05, *** p < 0.001). Protein was extracted from siRNA transfected HepG2 cells (condition as
in A). Western blots (B) were performed using 25 µg of whole cell extract and antibodies
targeted against PPAR, PPAR/ and PPAR (sc-9000, sc-7197, sc-7196, respectively,
Santa Cruz Biotechnologies). Cellular proteins were separated using 12 % SDS
polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The blotted
proteins were detected by using 1:15000 dilution of DyLight800 conjugated goat anti-rabbit
IgG (Pierce) and OdysseyTM infra-red imaging system (LI-COR Biosciences, Lincoln,
Nebraska, USA).