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 / Folds of Regulation / Cell type / Conditions / ComparisonBIRC3 / Microarray Assay / 1.4 + 4.6 / NS shRNA expressing ARPE-19 cells / OS / NS
3.2 + 1.8 / OS +NPD1
1.5 + 0.6 / 15LOX1 shRNA expressing ARPE-19 cells / OS / P < 0.05
5.1 + 2.1 / OS +NPD1
RT-PCR Array / 4 + 0.2 / ARPE-19 cells TNFR1 shRNA / OS vs OS+ NPD1 / P < 0.05
4.2 + 0.4 / ARPE-19 cells NS shRNA / OS / NS
5.7 + 0.5 / OS +NPD1
Real-Time PCR / 10.1 + 3.6 / A ARPE-19 cells TNFR1 shRNA / OS / P < 0.05
18.5 + 0.7 / OS + NPD1
3.27+0.4 / ARPE-19 cells NS shRNA / OS / P < 0.05
10.45+2.5 / OS + NPD1
Western Blot / 2.8 + 0.4 / ARPE-19 cells / OS / P < 0.05
3.4 + 0.4 / OS + NPD1
BIRC1 / Real-Time PCR / 0.86 + 0.15 / ARPE-19 cells / OS / NS
0.56 + 0.14 / OS + NPD1
BIRC2 / 0.92 + 0.19 / OS / NS
0.87 + 0.14 / OS + NPD1
BIRC3 / 12.7 + 0.9
16.5 +0.6
BIRC4 / 0.35 + 0.1 / OS / NS
0.24 + 0.4 / OS + NPD1
BIRC5 / 1.39 + 0.22 / OS / P < 0.05
0.88 + 0.13 / OS + NPD1
BIRC6 / 0.72 + 0.16 / OS / NS
0.34 + 0.05 / OS + NPD1
BIRC7 / 1.21 + 0.19 / OS / P < 0.05
0.55 + 0.12 / OS + NPD1
BIRC8 / 2.27 + 0.35 / OS / P < 0.05
0.67 + 0.32 / OS + NPD1
Supplementary Table S2: BIRC3 promoter computational analysis.Computational analysis of the binding consensuses sequences contained in the human BIRC3 promoterfor 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…-201TGGAAATCCC / -196…-187
TGGGTTTGCC / -159…-149
GGTTATTACC / -148…-139
CTGGAGTTCC / -147…-138
TGGAGTTCCC / -124…-115
GGAAAGCACC
cREL / TRED / 3.92 / 4.5 / 4.1 / 4.87 / N/A / N/A
TFSEARCH / 100 / N/A / N/A / N/A / N/A / 87
TFBIND / 1.00 / N/A / 0.83 / 0.85 / N/A / 0.92
p65 / TRED / 6.43 / 3.67 / N/A / 3.42 / 7.53 / N/A
TFSEARCH / 97.4 / N/A / N/A / N/A / N/A / N/A
TFBIND / 0.815 / 0.82 / N/A / 0.91 / N/A / 0.85
P50 / TRED / 5.65 / 2.64 / N/A / 1.57 / 5.83 / N/A
TFSEARCH / 91.4 / N/A / N/A / N/A / N/A / N/A
TFBIND / 0.79 / N/A / N/A / 0.75 / N/A / 0.79
*Max. Score: 100; **Max. Score: 1.00
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.
cREL promoter sequences*Searching Engine / -580... -571
GGGGGTCCCC / -281...-272
GGGGATTTCT / -10...-5
CGGGAAATTCC
TRED / 3.06 / 5.75 / 8.69
TFBIND / N/A / 0.81 / 0.98
TFsearch / N/A (cREL); 88.3 (p50) / 86.8 / 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.
RelB promoter consensus sequences*Searching engine / -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-ĸB-driven luciferase assay (Figure 2e).
Sequence / Transcription factor / Score / Searching EngineGGGACTTTCC / cREL / 9.19 / TRED
P65/RelA / 10.48
P50 / 5.08
GGGACTTTCCGGGACTTTCC / Heterodimer p65/p50 / 97.5 / TFSearch
cREL / 95.9
Supplementary Table S6: List of primers used in the ChiP assay.
gene / Forward primer / Reverse primerBIRC3 Promoter / 5’-GGGCATATTGACCTTTTCCA-3’ / 5’-AAATCCCCACCCCTATCTGT-3’
Supplementary Table S7: List of primers used for Real-Time PCR using SYBR green.
gene / Forward primer / Reverse primerb actin / 5'-GGACTTCGAGCAAGAGATGG-3' / 5'- AGCACTGTGTTGGCGTACAG-3'
Hm18s / 5'-CGCGGTTCTATTTTGTTGGT-3' / 5'- AGTCGGCATCGTTTATGGTC-3'
GAPDH / 5'-GAGTCAACGGATTTGGTCGT-3' / 5'- TTGATTTTGGAGGGATCTCG-3'
BIRC1 / 5'-TTCTTGCCCTGAAAACTGCT-3' / 5'-CGTATTGGGAAGTGGATGCT-3'
BIRC2 / 5'-GGAGATGATCCATGGGTAGA-3' / 5'-ACAAACTCTTGGCCTTTCAT-3'
BIRC3 / 5'-TGTTGGGAATCTGGAGATGA-3' / 5'-CGGATGAACTCCTGTCCTTT-3'
BIRC4 / 5'-GGGTTTGTCTTGACCTGGAA-3’ / 5'-CTCCCAAAGTGCTGGGATTA-3’
BIRC5 / 5'-TCCCTGGCTCCTCTACTGTT-3' / 5'-TGTCTCCTCATCCACCTGAA-3'
BIRC6 / 5'-ACCCTTGCTTCCTCTTCTCA-3' / 5'-AACCACCAAGCCAATCTTTC-3'
BIRC7 / 5'-CTCTGCCTGTTCTGGACTGT-3' / 5'-CCAGGGAAAACCCACTTTAT-3'
BIRC8 / 5'-ATTCAAACATCTGGGAGCAA-3' / 5'-CAAAGCTTCTCCTCTTGCAG-3'
Crel / 5'-CAGGAGGAAGAGCAGTCGTC-3' / 5'- GCAGGAATCAATCCATTCAA-3'
RelA / 5'-GGTCCGCTGAAAGGACTCTT-3' / 5'-GAATTCCAGTACCTGCCAGA-3'
RelB / 5'-GAGGCCAGTCCTTCCACAC-3' / 5'-ATCCTTGGGGAGAGCAGC-3'
TNFR1 / 5'-GCCAGGAGAAACAGAACACC-3' / 5'- CTCAATCTGGGGTAGGCACA-3'