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Supplemental Figure 1. A. B. Inhibitors concentration (µM). - PowerPoint PPT Presentation
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Supplemental Figure 1
Piceatannol 1 5 10 30 50 100
SP600125 0,1 1 2 5 10 20
ER-27319 1 5 10 30 50 100
Inhibitors concentration (µM)
A
B
NI HIV0
20
40
60
80
100
120
140
160MockPiceatannolSP600125ER-27319
% o
f cel
l via
bilit
y
NT
AMD3100
GF109203
wortmannin
PD98059 PDTC
SP600125
SB203580
ER-273190
20
40
60
80
100
120MockHIV
% o
f cel
l via
bilit
y
Supplemental Figure 1: B cell survival (A) B cells were pre-treated with kinase inhibitors (GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM) and ER-27319 (SYK, 50µM)) or with AMD3100 (100µM) for 4h before HIV-1 stimulation. 24h post-stimulation, percentages of cell viability were analyzed with 7AAD by flow cytometry. One representative experiment from 4 is represented (average+SEM). (B) B cells were pre-treated with increasing concentrations of JNK and SYK inhibitors (SP600125 (JNK, 0,1µM to 20µM); ER-27319 and Piceatannol (SYK 1µM to 100µM)) for 4h before HIV-1 stimulation. 24h post-stimulation, percentages of cell viability were analyzed with 7AAD by flow cytometry. Results from one representative experiment from two. Doted line represent the threshold of cell viability determined at 80% in comparison to Mock-NI condition.
Supplemental Figure 2
Supplemental Figure 2: Cell activation and inhibition of activation by kinases inhibitors. B cells were pre-treated with kinase inhibitors (GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM); ER-27319 (SYK, 50µM); Piceatannol (SYK, 50µM)) and AMD3100 (100µM) for 4h before CD40L/IL-4 stimulation. 24h post-stimulation, iMFI of cells expressing CD71, CD69 or CD86 were followed by flow cytometry. Average of iMFI + SEM of minimum 4 experiments for each conditions (4 to 6 different donors for the NA (non-activated) conditions and 6 to 10 donors for the CD40L/IL-4 conditions). NA; Non-activated (*=p<0,05 when comparing NT-NA condition with kinase inhibitor-NA and #=p<0,05 when comparing NT-CD40L/IL-4 condition and kinases inhibitor-CD40L/IL-4). NT: non-treated.
01020304050607080 NA
CD40L/IL-4iM
FI (C
D71
)
010002000300040005000600070008000 NA
CD40L/IL-4
iMFI
(CD
69)
NT
GF109203
PD98059
SP600125
ER-27319 0
100200300400500600 NA
CD40L/IL-4
iMFI
(CD
86)
*
#
#
#
*
* *
##
# #
#
**
*
Supplemental Figure 3
0.1
1
10Non-activated
AID
mRN
A (F
old
chan
ge, l
og)
0.1
1
10
100CD40L/IL-4
AID
mRN
A (F
old
chan
ge, l
og)
*
**
NT
GF1
0920
3
wor
tman
nin
PD98
059
PDTC
SP60
0125
SB20
3580
ER-2
7319
Pice
atan
nol
CD71
CD69
CD86
0
2
4
6
8
10
12
NT-NAHIVCD40L/IL-4HIV/CD40L/IL-4
iMFI
(fol
d ch
ange
)
CB
*##
*#
A
NT-NA
CD40L/IL-4
0
5
10
15
20
AID
mRN
A (F
old
chan
ge)
Supplemental Figure 3: AID mRNA expression in CD40L/IL-4 treated B cells. (A) B cells were pre-treated with kinase inhibitors; GF109203 (PKC, 0,5µM); wortmannin (PI3K, 100nM); PD98059 (MEK1, 5µM); PDTC (NF-κB, 5µM); SP600125 (JNK, 5µM); SB203580 (p38, 5µM), ER-27319 (SYK, 50µM) or Piceatannol (SYK, 50µM) for 4h before CD40L/IL-4 stimulation or cells were kept non-activated as control. Two days after stimulation, total mRNA was extracted and AID mRNA were quantified by real-time PCR. Fold changes were calculated in comparison to non treated (NT) condition that was considered a fold change of 1. Each black (Non-activated conditions) or grey (CD40L/IL-4 stimulated conditions) plot represents one donor. Black bar represents the average of AID mRNA fold change . (B) B cells were stimulated with HIV, CD40L/IL-4 or with HIV/CD40L/IL-4 for 24h. iMFI of cells expressing CD71, CD69 or CD86 were followed by flow cytometry. Fold changes were calculated in comparison to NT-NA (Non-Activated) condition that is considered a fold change of 1. Average of iMFI + SEM of minimum 4 independent experiments for each conditions (4 or 5 different donors regarding the condition). #=p<0,05 when comparing HIV condition with HIV/CD40L/IL-4 and *=p<0,05 when comparing CD40L/IL-4 condition with HIV/CD40L/IL-4 (C) B cells were stimulated with HIV, CD40L/IL-4 or with HIV/CD40L/IL-4. Two days after stimulation, total mRNA was extracted and AID mRNA were quantified by real-time PCR. Fold changes were calculated in comparison to NT-NA condition that is considered a fold change of 1. Average + standard deviation of 2 independent experiments is shown.
Supplemental Figure 4
V(D)Jµ δ γ2
IgMGermline IH-CH transcription
AID
V(D)Jγ2
IgG2Productive (VDJ-CH) Ig transcript
Excised DNA segment=circle
transcript
CSR
δµ
Supplemental Figure 4: Class Switch Recombination (CSR). Mechanism of class switch recombination that allows isotype selection in mature activated B cells. This mechanism produces a DNA recombinant of the constant region of the immunoglobulin heavy chain and an excised DNA segment.