Supplementary information
Material and Methods
RT-PCR analysis of biopsy samples
Total RNA was extracted from frozen biopsy specimens with Qiagen RNeasy isolation system
(Qiagen, Hilden, Germany), treated with RNase-free DNase I (Qiagen) and quantified by the
OD260/280 ratio using the Nanodrop spectrophotometer ND-1000 (Labtech). The RNA quality
was evaluated on Agilent 2100 bioanalyser using the RNA 6000 Labchip kit (Agilent
Technologies, Palo Alto, CA). For cDNA synthesis total RNA (2 µg) was reverse transcribed
with the Super Script III First-strand synthesis kit (Invitrogen) according to the manufacturer's
instructions, using oligo(dT) or with high-capacity cDNA reverse transcription kit (Applied
Biosystems, Foster City, CA), Amplification was performed using TaqDNA polymerase
(Invitrogen) and specific primers as previously reported [17, 18]. Transcripts of NGF, BDNF,
TrkA, TrkB, p75NTR and sortilin were obtained using UnoCycler (VWR, Fontenay-sous-Bois,
France). Thirty-five PCR cycles were performed (94°C for 30 s, 58°C or 60°C for 30 s as
previously described in Bellanger et al, 2011, 72°C for 45 s, and an extension step of 72°C for
7 min).
Real-time quantitative PCR analysis
Total RNA (1 µg) was used as template for cDNA synthesis (Applied Biosystems, Foster City,
CA) and the cDNAs were used for quantitative real-time PCR analysis using TaqMan probes,
labeled with 5′ reporter dye 6FAM and the 3′ Minor Groove Binder-Non-Fluorescent Quencher
(MGB-NFQ) (Applied Biosystems). Experiments were performed in duplicate in at least three
independent experiments and PCR runs were performed and analysed on an Applied
Biosystems StepOnePlus Real-Time (Applied Biosystems), with a two-step PCR protocol
(95°C for 20 s followed by 40 cycles of 95°C for 3 s and 60°C for 30 s). Each reaction was
assessed using 400 nM of the forward and reverse primers, 200 nM labeled probe, and 100 ng
cDNA template (RNA-cDNA equivalent) mixed with TaqMan Fast-Univ Master Mix (Applied
Biosystems). BDNF, full-length (TrkB145) and truncated (TrkB95) TrkB, and p75NTR primers
were purchased from Lifetechnologie. The sequences of primers and TaqMan probes are shown
in Table S1. Data were normalized to Hypoxanthine phosphoribosyltransferase (HPRT)
housekeeping gene as an internal control (Predesigned Human HPRT Primer/Probe Mix;
Applied Biosystems). The quantitative PCR efficiency was assessed using regression curve
performed with cDNA serial dilutions samples (400, 200, 100, 50, and 25 ng cDNA) (Peirson
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et al. 2003) (PMID: 12853650). The comparative Ct method was used for relative
quantification of gene expression.
Transfections and lentivirus infections
293T cells were transfected with pLKO control vector or pLKO_shp75NTR vector (Sigma
SHCLND-NM_002507) following standard procedures. 48 hours after transfection, media
containing lentiviral particles (control or shp75NTR) was collected and used for DLBCL cell
lines infection. 48 hours after infection, cells were selected with 5 µg/ml of puromycin. p75NTR
interference was confirmed by western blotting and pRT-PCR.
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2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
NGF
BDNF
TrkA
TrkB95
TrkB145
p75NTR
Sortilin
Actin/GAPDH
1: non-GCB
2: non-GCB
3: non-GCB
4: GCB
5: non-GCB
6: GCB
7: GCB
8: GCB
9: GCB
10: GCB
11: GCB
12: GCB
13: non-GCB
14: non-GCB
15: GCB
16: GCB
Figure S1. mRNA expression of neurotrophins and their receptors in DLBCLs:
comparison with cell-of-origin classification. RT-PCR analysis was performed on 6 non GCB
and 10 GCB DLBCL patient biopsies using specific primers to NGF, BDNF, TrkB (truncated
and full-length receptors), TrkA, p75NTR and sortilin. Constitutively expressed actin or GAPDH
(patient 10 to 16) is a control of PCR efficiency.
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Figure S2. Modulation of BDNF, TrkB and p75NTR transcript expressions in DLBCL cell
lines. Quantification polymerase chain reaction assay was performed to measure the mRNA
relative levels of BDNF, full-length (Trk145) and truncated (Trk95) TrkB and p75NTR in GCB
(SUDHL4 and SUDHL6) versus ABC (OCI-LY3 and OCI-LY10) cell lines. Gene expressions
were compared to the SUDHL4 cell line mRNA levels and normalized to HPRT housekeeping
gene mRNA levels. Histograms represent relative quantification means ± SEM. “p” values
were evaluated by comparing to the SUDHL4 mRNA levels by One-Way ANOVA.
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A
B
C
sh_C
1
sh_C
sh_p75
0.54
sh_C
sh_p75
sh_p75
p75NTR
p75NTR
p75NTR
Actin
Actin
Actin
p75/Actin
1
0.32
p75/Actin
1
0.47
p75/Actin
Figure S3. Inhibition of p75NTR expression increases the rituximab sensitivity of DLBCL
cell lines. Human GCB-like (SUDHL4 and SUDHL6) and ABC-like (OCI-LY10) DLBCL cell
lines were transduced with lentivirus carrying shRNA against p75NTR (shp75) or empty vector
(sh_C). (A) Viability was assessed with or without rituximab (1 µg/ml) 48 hours after treatment
by using XTT method. Histograms show the quantification of at least three independent
experiments performed in triplicates each one. (B) Relative levels of p75NTR were evaluated by
qRT-PCR for the three DLBCL cell lines. (C) Protein levels for p75NTR were analysed by
western blotting in DLBCL cell lysates for controls (sh_C) or p75NTR reduced conditions
(sh_p75). Actin was used as loading control. Student t test was used to determine significance,
*: p<0.05; and ***: p<0.001 when compared to controls.
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Table S1. Sequences of primer pairs and probes used for real-time RT-PCR
Sequences
GenBank
references
Forward
GGCTATGTGGAGTTGGCATT
NM_170735.5
Reverse
CAAAACGAAGGCCTCTGAAG
Probe
ATTTCTGAGTGGCCATCCCAAGGTCTAG
Forward
CTGGTGAAAATCGGGGACT
Names
BDNF
TrkB-FL
Full lenght Reverse
AGGAAATTCACGACGGAAAG
(TrkB145)
Probe
TGTACAGCACTGACTACTACAGGGTCGGTG
TrkB-T1
Forward
AAGATCCCACTGGATGGGTA
Truncated
Reverse
GGAAGTGCTGCTTATCTGGG
(TrkB95)
Probe
ATAAAGGAAAAGACAGAGAAAGGGGCTGTG
P75NTR
Forward
CACCACCGACAACCTCATC
Reverse
AAGCAGAACAAGCAAGGAGC
Probe
CCTACATAGCCTTCAAGAGGTGGAACAGCT
NM_006180.3
NM_00100709
7.1
NM_002507.3
Table S2. Correlation of combined immunohistochemical scores and DLBCL subtype
NGF
TrkB
BDNF
0.353
p=0.01
p75NTR
0.377
p=0.006
Mum1/IRF4
0.369
p=0.008
GCB
versus non-GCB phenotype
0.280
p=0.04
Correlation coefficient and p-value as determined by the Spearman rank correlation test.
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Table S3. Phenotype characteristics and NGF status of patients with DLBCL
NGF expression
N
MUM1/IRF4
Negative
positive
Low1
High
21
30
11
10
6
24
p=0.03
NGF staining scores of 0-2 are denoted as “low” and scores of 3-4 as “high”
N: number of patients - p -value as determined by the Fisher’s exact test.
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