Supplementary Table S1: BIRC mRNA and protein expression. Expression of BIRC3 and
other members of the family upon oxidative stress when treated with NPD1 in different cell types
including ARPE-19 cells deficient in TNFR1 and 15-LOX-1. TNFR1-d cells present a decreased
TNFR1/TNF-α cytotoxic pathway. 15-LOX-1 silenced cells depict low levels of NPD1.Summary
of BIRC expression assays, which include Microarray, Real-time PCR-Array, Real-Time PCR
and Western blot performed for this report. Folds of regulation and statistical treatment of the
comparisons are detailed.
Gene ID
Assay
Microarray Assay
BIRC3
RT-PCR Array
Real-Time PCR
Western Blot
BIRC1
BIRC2
BIRC3
BIRC4
Real-Time PCR
BIRC5
BIRC6
BIRC7
BIRC8
Folds of Regulation
1.4 + 4.6
3.2 + 1.8
1.5 + 0.6
5.1 + 2.1
4 + 0.2
4.2 + 0.4
5.7 + 0.5
10.1 + 3.6
18.5 + 0.7
3.27+0.4
10.45+2.5
2.8 + 0.4
3.4 + 0.4
0.86 + 0.15
0.56 + 0.14
0.92 + 0.19
0.87 + 0.14
12.7 + 0.9
16.5 + 0.6
0.35 + 0.1
0.24 + 0.4
1.39 + 0.22
0.88 + 0.13
0.72 + 0.16
0.34 + 0.05
1.21 + 0.19
0.55 + 0.12
2.27 + 0.35
0.67 + 0.32
Cell type
Conditions
NS shRNA expressing ARPE-19 cells
15LOX1 shRNA expressing ARPE-19 cells
ARPE-19 cells TNFR1 shRNA
ARPE-19 cells NS shRNA
A ARPE-19 cells TNFR1 shRNA
ARPE-19 cells NS shRNA
ARPE-19 cells
OS
OS +NPD1
OS
OS +NPD1
OS vs OS+ NPD1
OS
OS +NPD1
OS
OS + NPD1
OS
OS + NPD1
OS
OS + NPD1
OS
OS + NPD1
OS
OS + NPD1
ARPE-19 cells
OS
OS + NPD1
OS
OS + NPD1
OS
OS + NPD1
OS
OS + NPD1
OS
OS + NPD1
Comparison
NS
P < 0.05
P < 0.05
NS
P < 0.05
P < 0.05
P < 0.05
NS
NS
NS
P < 0.05
NS
P < 0.05
P < 0.05
Supplementary Table S2: BIRC3 promoter computational analysis. Computational analysis
of the binding consensuses sequences contained in the human BIRC3 promoter for 3
components of NF-ĸB. In silico promoter analysis performed by three different searching
engines: TRED using Jaspar Matrix; TFSearch and TFbind using TRANSFACT Matrix. The
maximum scores for TFSearch and TFbind are marked with an asterisk and displayed at the
bottom of the table. The table depicts the score obtained for each NF-ĸB element: cREL,
p65/RelB and p50 (column) in the corresponding binding site (row). The best score values were
obtained for cREL in the site that goes from -211 to -201 bp within the fragment that showed
NPD1 activation.
Position
-211…-201
TGGAAATCCC
TRED
3.92
cREL
TFSEARCH
100
TFBIND
1.00
TRED
6.43
p65
TFSEARCH
97.4
TFBIND
0.815
TRED
5.65
P50
TFSEARCH
91.4
TFBIND
0.79
*Max. Score: 100; **Max. Score: 1.00
-196…-187
TGGGTTTGCC
-159…-149
GGTTATTACC
-148…-139
CTGGAGTTCC
-147…-138
TGGAGTTCCC
-124…-115
GGAAAGCACC
4.5
N/A
N/A
3.67
N/A
0.82
2.64
N/A
N/A
4.1
N/A
0.83
N/A
N/A
N/A
N/A
N/A
N/A
4.87
N/A
0.85
3.42
N/A
0.91
1.57
N/A
0.75
N/A
N/A
N/A
7.53
N/A
N/A
5.83
N/A
N/A
N/A
87
0.92
N/A
N/A
0.85
N/A
N/A
0.79
Supplementary Table S3: cREL promoter computational analysis. Computational analysis
of consensus sequences found in cREL promoter. The table depicts the scores obtained for
cREL binding for each consensus sequence.
Searching
Engine
TRED
TFBIND
TFsearch
-580... -571
GGGGGTCCCC
3.06
N/A
N/A (cREL); 88.3 (p50)
cREL promoter sequences*
-281...-272
GGGGATTTCT
5.75
0.81
86.8
-10...-5
CGGGAAATTCC
8.69
0.98
92.6
Supplementary Table S4: RelB promoter computational analysis. Computational analysis of
the consensus sequences found in the RelB promoter. The table depicts the scores obtained for
the binding of cREL to each consensus sequence.
Searching engine
RelB promoter consensus sequences*
-534… -525 GGAGGTTTCC -247…- 238 GGGGTTTTCC
-175… - 166 GGGAATTCC
TRED
6.77
9.27
9.26
TFBind
0.91
0.98
0.98
Tfsearch
86
97.5
95.9
Supplementary Table S5: Computational analysis of binding affinity of NF-ĸB
components to the tandem repeated consensus sequences contained in the NF-ĸBdriven luciferase assay (Figure 2e).
Sequence
GGGACTTTCC
GGGACTTTCCGGGACTTTCC
Transcription factor
cREL
P65/RelA
P50
Heterodimer p65/p50
cREL
Score
9.19
10.48
5.08
97.5
95.9
Searching Engine
TRED
TFSearch
Supplementary Table S6: List of primers used in the ChiP assay.
gene
BIRC3 Promoter
Forward primer
5’-GGGCATATTGACCTTTTCCA-3’
Reverse primer
5’-AAATCCCCACCCCTATCTGT-3’
Supplementary Table S7: List of primers used for Real-Time PCR using SYBR green.
gene
b actin
Hm18s
GAPDH
BIRC1
BIRC2
BIRC3
BIRC4
BIRC5
BIRC6
BIRC7
BIRC8
Crel
RelA
RelB
TNFR1
Forward primer
5'-GGACTTCGAGCAAGAGATGG-3'
5'-CGCGGTTCTATTTTGTTGGT-3'
5'-GAGTCAACGGATTTGGTCGT-3'
5'-TTCTTGCCCTGAAAACTGCT-3'
5'-GGAGATGATCCATGGGTAGA-3'
5'-TGTTGGGAATCTGGAGATGA-3'
5'-GGGTTTGTCTTGACCTGGAA-3’
5'-TCCCTGGCTCCTCTACTGTT-3'
5'-ACCCTTGCTTCCTCTTCTCA-3'
5'-CTCTGCCTGTTCTGGACTGT-3'
5'-ATTCAAACATCTGGGAGCAA-3'
5'-CAGGAGGAAGAGCAGTCGTC-3'
5'-GGTCCGCTGAAAGGACTCTT-3'
5'-GAGGCCAGTCCTTCCACAC-3'
5'-GCCAGGAGAAACAGAACACC-3'
Reverse primer
5'- AGCACTGTGTTGGCGTACAG-3'
5'- AGTCGGCATCGTTTATGGTC-3'
5'- TTGATTTTGGAGGGATCTCG-3'
5'-CGTATTGGGAAGTGGATGCT-3'
5'-ACAAACTCTTGGCCTTTCAT-3'
5'-CGGATGAACTCCTGTCCTTT-3'
5'-CTCCCAAAGTGCTGGGATTA-3’
5'-TGTCTCCTCATCCACCTGAA-3'
5'-AACCACCAAGCCAATCTTTC-3'
5'-CCAGGGAAAACCCACTTTAT-3'
5'-CAAAGCTTCTCCTCTTGCAG-3'
5'- GCAGGAATCAATCCATTCAA-3'
5'-GAATTCCAGTACCTGCCAGA-3'
5'-ATCCTTGGGGAGAGCAGC-3'
5'- CTCAATCTGGGGTAGGCACA-3'