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Description of Supplementary Files File Name: Supplementary Information Description: Supplementary Figures and Supplementary Table File Name: Supplementary Data 1 Description: List of genes differently expressed among the classes passing the cut‐off value in T cells. The genes are tabulated along with parametric p‐value, FDR, the geometric mean of intensities, fold change and links to major annotation sources. File Name: Supplementary Data 2 Description: List of genes differently expressed among the classes passing the cut‐off value in Monocytes. The genes are Pag. 5 di 5 tabulated along with parametric p‐value, FDR, the geometric mean of intensities, fold change and links to major annotation sources. File Name: Supplementary Data 3 Description: List of the different gene sets identified in T cell, performing the gene set comparison tool in BRB Array‐Tools as a scoring test to assign the functional category definitions according to the Gene Ontology Database. File Name: Supplementary Data 4 Description: List of the different gene sets identified in Monocytes, performing the gene set comparison tool in BRB ArrayTools as a scoring test to assign the functional category definitions according to the Gene Ontology Database. File Name: Peer Review File
Supplementusing A) AFdimension di
and thicknes=500nm).
(E
Summary of number of pa
tary Figure 1FM, followed istribution (Sc
ss distributionE) Raman spe
f the characteriarticles analyz
1. Physicocheby lateral dimcale Barr =50
s (Scale Barr ctra of GO (r
ization. Data azed.
emical charamension and t00nm). GONH
=500nm); D)red) and GON
are presented
acterization othickness distH
2 characteriz
) TEM microNH
2 (blue). F
as mean ± SD
of GO and Gtributions (Scazation using C
graph with la) FT-IR spect
D. Microscopy
GONH2. GO
ale Barr =500C) AFM, follo
teral dimensiotra of GO (re
y data are acco
structural ch0nm); B) TEMowed by later
on distributioed) and GONH
ompanied by
haracterizationM with lateralral dimension
n (Scale BarrH
2 (blue). G)
the range and
n l n
r )
d
Supplementary Figure 2. Summary of SPADE analysis. SPADE tree plots of untreated samples (CTRL) for significantly expressed cytokines (IL6, TNFα and MIP1β).
Supplementary Figure 3. Single-cell characterization on gated T lymphocyte subpopulations in GO treated samples (viSNE analysis). Plots show the use of viSNE to obtain a comprehensive single cell view and to distinguish the T cell subpopulations in the GO treated cells. Plots show expression of 9 proteins including cytokines.
cells
Monocytes
mDCs
pDCs
CD16+ NK Cells
CD16- NK Cells
Activated T HelperNaive T Helper
Memory T Helper
Naive T Killer
Memory T killer
Effector T Killer
Activated T killer
mory B cells
Plasma B Cells
Naive B cells
Helper
0
3.24
Monocytes
mDCs
pDCs
CD16+ NK Cells
CD16- NK Cells
Activated T HelperNaive T Helper
Memory T Helper
Naive T Killer
Memory T killer
Effector T Killer
Activated T killer
Memory B cells
Plasma B Cells
Naive B cells
Effector T Helper
,0
3.05
Monocytes
mDCs
pDCs
CD16+ NK Cells
CD16- NK Cells
Activated T HelperNaive T Helper
Memory T Helper
Naive T Killer
Memory T killer
Effector T Killer
Activated T killer
Memory B cells
Plasma B Cells
Naive B cells
Effector T Helper
1 cell9,398.08 cells0
3.24
1 cell9,398.08 cel0
3.05
1 cell9,398.08 cells0
2.77
IL6 TNFα MIP1β
NaïveT Helper
pDCs
Monocytes
Effector T Helper
Activated T Helper
Memory T Helper
Naïve T Cytotoxic
Effector T Cytotoxic
Activated T Cytotoxic
Memory T Cytotoxic
Plasma B cells
Naïve B cells
mDCs
Memory B cells
NaïveT Helper
pDCs
Monocytes
EffectorT Helper
Activated T Helper
Memory T Helper
Naïve T Cytotoxic
Effector T Cytotoxic
Activated T Cytotoxic
Memory T Cytotoxic
Plasma B cells
Naïve B cells
mDCs
Memory B cells
NaïveT Helper
pDCs
Monocytes
EffectorT Helper
Activated T Helper
Memory T Helper
Naïve T Cytotoxic
Effector T Cytotoxic
Memory T Cytotoxic
Plasma B cells
Naïve B cells
mDCs
Memory B cells
Activated T Cytotoxic
3.15
Lymphocytes T Helper
Lymphocytes T Cytotoxic
Activated
Memory
Effector
Naive
CD27 CD45RA CD38 HLADR
IFNg IL2 IL4 IL5 TNFa
CD27 CD45RA CD38 HLADR
Activated
Memory Effector
Naive
IFNg IL2 IL4 IL5 TNFa
Supplementary Figure 4. Single-cell characterization of gated T lymphocyte subpopulations in GONH
2 treated
samples (viSNE analysis). Plots show the use of viSNE to obtain a comprehensive single cell view and to distinguish the T cell subpopulations in the GONH
2 treated cells. Plots show expression of 9 proteins including cytokines.
Lymphocytes T Helper
Lymphocytes T Cytotoxic
CD27 CD45RA CD38 HLADR
Activated
Memory
Effector
Naive
IFNg IL2 IL4 IL5 TNFa
CD27 CD45RA CD38 HLADR
Activated
Memory Effector
Naive
IFNg IL2 IL4 IL5 TNFa
Supplementary Figure 5. Single-cell characterization of GO and GONH
2 on gated B cells (viSNE analysis). Plots
show the use of viSNE to obtain a comprehensive single-cell view and to distinguish the B cell subpopulations in the GO and GONH
2 treated cells. Plots show expression of the 7 proteins used to distinguish the single population
including cytokines.
B Cells
GONH2
GO
HLADR CD27 CD38
Memory
Plasma Naive
IL2 IL4 IL5 TNFa
CD38
Memory
Plasma Naive
HLADR CD27
IL2 IL4 IL5 TNFa
Supplementary Figure 6. Single-cell characterization of gated NK cells and monocytes in GO and GONH
2
treated samples (viSNE analysis). Plots show the use of viSNE to obtain a comprehensive single-cell view and to distinguish the NK cells and monocytes in GO and GONH
2 treated cells. Plots show expression of 9 proteins including
cytokines for NK cells and 6 for monocytes.
NK Cells
Monocytes
CD16 HLADR CD27 CD38
CD14 CD16 HLADR CD38 TNFa IL6
IL4 IL5 TNFa
GO
NK Cells
Monocytes
GONH2
CD14 CD16 HLADR CD38 TNFa IL6
CD16 HLADR CD27 CD38
IL4 IL5 TNFa
Supplement
50, 100 µg·mof human RBthe damagedpositive conttriplicate (**multiple com
tary Figure 7
mL-1) with redBCs treated wd RBCs. PBStrol, respectiv
**= P value <mparison test. W
7. Hemolysis
d blood cells (with the differeS mixed with vely. PBS alo
<0.0001, ns= PWhere not spe
analysis. GO
(RBCs) at 25°ent GOs. The RBCs (-) an
one was usedP value >0.05ecified the P v
O and GONH2
°C for 2 h. a)red color of t
nd ultrapure wd as additiona5 Statistical anvalue is intend
2 were incuba
Sample absothe solution iswater mixed wal control (Wnalysis perforded vs. Ctrl).
ted (at increa
rbance measus due to the rewith RBCs (+
W). The experimed by two-w
asing concentr
ured at 570 nmelease of hem+) served as iments were way ANOVA
rations: 5, 25,
m. b) Picturesmoglobin from
negative andperformed in
A and Tukey’s
,
s m d n s
Supplementsignificance
identified usi
tary Figure 8level [Fisher
ing IPA softw
. Ingenuity pexact test -lo
ware.
athways analog (p-value)]
lysis. Top 20 modulated b
canonical patby GO and G
thways and Z-GONH
2 in a)
-score rankingT-cells and b
g according tob) monocytes
o s
Supplementmodulated inGONH2 in T
tary Figure 9.n GO sample. T-cells.
. Gene set enrb) Gene onto
richment witlogy visualiza
th Gene Ontoationheatmap
ology and IPAof OXPHOS p
A analysis. a) pathway after
OXPHOS par treatment wit
thway found th GO and
Supplementwith GONH
2
tary Figure 1
2.
10. Significannt canonical pathways. DDC maturationn pathway in monocytes af
fter treatmentt
SupplementGONH
2 (refe
tary Figure 1er to Supplem
11. Significanentary Figure
nt canonical p10 legend).
pathways. IFFN signaling ppathway in TT-cells after tr
reatment withh
Supplement
triplicate by measure of f
value <0.000
tary Figure 1
quantitative fold changes b
04 Statistical a
12. Microarr
real-time PCbetween GO a
analysis perfor
ray data vali
R. Histogramand GONH
2 a
rmed by one-w
idation in mo
ms show residand controls (a
way ANOVA
onocytes cell
dual mRNA lalways expres
A test with Bon
line. mRNA
level expressessed as 1) (**=
nferroni correc
A levels were
ed as 2-ΔΔct
t= P value < 0
ction).
measured in
that gives the0.001, ***= P
n
e P
Supplementthe identificaThe gating wwas performe
tary Figure 1ation of the d
was performeded first using
13. Gating strdifferent immud following thIr 191 Di(DN
rategy of diffune cell subpohe Fluidigm inNA1) and Ir 19
ferent immunopulations is nstruction of th93 Di (DNA2)
ne cell subposhown for thehe Maxpar pa), LD stands fo
opulations. The control samanel used. Theor CIS.
he used gatinmple as a conte cell events d
g strategy forour dot plots.
discrimination
r .
n
Supplementthree times w
confirming tcomparison t
tary Table 1. with GO and G
the robustnestest of every a
SPADE robuGONH
2 sampl
ss of the SPAalgorithm run.
ustness evalules. The numb
ADE data we
uation amongber of major c
ere performed
g three indepcell population
d using a tw
endent runs. n events are re
o-way ANOV
SPADE analeported. Statis
VA and Tuk
lysis were runstical analyses
ey’s multiple
n s
e