Supplementary Information
A novel LSD1 inhibitor T-3775440 disrupts GFI1B-containing complex leading to
transdifferentiation and impaired growth of AML cells
Yoshinori Ishikawa1, Kanae Gamo1, Masato Yabuki1, Shinji Takagi1, Kosei Toyoshima1,
Kazuhide Nakayama1, Akiko Nakayama1, Megumi Morimoto1, Hitoshi Miyashita1, Ryo
Dairiki1, Yukiko Hikichi1, Naoki Tomita1, Daisuke Tomita1, Shinichi Imamura1, Misa
Iwatani2, Yusuke Kamada2, Satoru Matsumoto3, Ryujiro Hara1, Toshiyuki Nomura1,
Ken Tsuchida1, and Kazuhide Nakamura1
Supplementary Materials and Methods
Expression and purification of human LSD1/CoREST complex
The expression plasmids pET15SM/SUMO-hLSD1 (172–852) and pRH8/His-hCoREST (286–
482) were co-transfected into E. coli BL21 (DE3) competent cells. Transformed cells were
grown in modified M9 medium supplemented with 1% yeast extract. Protein expression was
induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells from a 3-L culture
were lysed by sonication, and His-SUMO-hLSD1 (172–852) and His-hCoREST (286–482)
were purified using a Ni-NTA superflow cartridge (QIAGEN, Hilden, Germany) and HiLoad
26/60 Superdex 200 pg (GE Healthcare, Little Chalfont, UK). The His-SUMO tag at the
N-terminal of hLSD1(172–852) was cleaved using SUMO protease (LifeSensors, Malvern, PA,
USA); subsequently, reverse NiNTA chromatography was used to remove SUMO protease and
cleave His-SUMO tags. Human LSD1 (172–852) and His-hCoREST (286–482) complex were
further purified with HiLoad 26/60 Superdex 200 pg equilibrated using phosphate-buffered
saline (PBS) containing 150 mM NaCl and 5% (V/V) glycerol. Protein concentrations were
measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), using
bovine serum albumin (BSA) as a protein standard.
LSD1 enzyme kinetic assay for determination of kinact/KI values
LSD1 enzyme activity was measured using a peroxidase-coupled assay. Various compound
concentrations were incubated with 500 µM mono-methylated Lys 4 on a histone H3 peptide
(synthesized by SCRUM Inc., Tokyo, Japan) in assay buffer (50 mM Tris-HCl (pH8.0), 0.1%
BSA). After adding 20 µg/mL horseradish peroxidase and 50 µM AmplexRed reagent (Life
Technologies, Carlsbad, CA, USA), the enzyme reaction was initiated by incubation with 20 nM
recombinant LSD1/CoREST. Hydrogen peroxide production during the LSD1 amine oxidase
1
reaction was monitored by AmplexRed reagent. Fluorescence signals of the resultant product
resorufin were excited at 570 nm and detected at 585 nm on a Spectra Max device (Molecular
Devices, Sunnyvale, CA, USA).
Because the compounds were irreversible inhibitors, their progress curves were fitted
to the equation 1 (below) using GraphPad Prism 5 Software (GraphPad Software, Inc., San
Diego, CA, USA). Fitted data were derived from the signals produced during the enzyme–
compound reactions, and the fluorescent signals of the compounds not subjected to enzymatic
reactions were subtracted as background from the total reaction signals.
y = y 0 + (vi/kobs) [1 − e (−kobs*t )],
(1)
where vi represents the initial velocity and kobs or t demonstrates the pseudo-first order rate
constant or time, respectively. y0 is the product when t is 0. The estimated kobs values were
replotted as a function of the inhibitor concentration, and kinact or KI values were calculated
using equation 2 (below). The calculated Km value for the histone H3 peptide substrate was 20
µM.
kobs = kinact/[1 + (KI /[I]) (1 + [S]/Km)]
(2)
In vitro LSD1 demethylation assay
The HTRF detection system (Cisbio, Codolet, France) was used to estimate in vitro LSD1
demethylation activity. The reaction buffer consisted of 50 mM Tris-HCl (pH8.0), 1 mM DTT,
and 0.01% BSA. For the demethylation reaction, 25 nM recombinant LSD1/CoREST and 1 μM
biotinylated
histone
H3K4
monomethylated
peptide
[ART(mK)QTARKSTGGKAPRKQLAGGK-Biotin] (SCRUM Inc.) were identified as the
optimal concentrations. The enzyme and compounds were incubated for 60 min prior to starting
the demethylation reaction. The reaction was initiated by adding a biotinylated histone H3K4
monomethylated
peptide,
and
terminated
after
15
min
by
adding
1
mM
trans-2-Phenylcyclopropylamine (2-PCPA). The demethylated peptide was detected using a
cryptate-labeled anti-histone H3 antibody and streptavidin-Xlent! (Cisbio), which were diluted
in detection buffer containing 800 mM potassium fluoride (KF) and 0.1% BSA. The anti-histone
H3 antibody was purchased from Wako Pure Chemical, Co. (Osaka, Japan), and cryptate
labeling of the antibody was done by Cisbio. After incubation at room temperature for 1 h, the
plates were read using an EnVision 2102 Multilabel Reader (PerkinElmer, Waltham, MA, USA).
We set the total reaction without enzyme as 100% inhibitory activity, and the total reaction as
0% inhibitory activity. Curve fittings and calculations of IC50 values were performed using the
program XLfit, ver. 5 (ID Business Solutions Ltd., London, UK) with the maximum and
minimum of the curve constrained to 100 and 0, respectively.
2
In vitro MAO-A /B enzyme assay
MAO-A/B enzyme activity was measured with a MAO-Glo™ Assay (Promega, Madison, WI,
USA) according to the manufacturer's instructions. MAO-A and MAO-B were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Briefly, a test compound was mixed with a reaction
solution containing MAO-A or MAO-B enzyme for 10 min, after which MAO substrate was
added to start the reaction. After a 60-min reaction at room temperature, Luciferin Detection
Reagent (Promega) was added to terminate the reaction. Luminescence was then measured
using the Envision device (PerkinElmer). We defined the luminescent signals of the reaction
without enzyme as 100% inhibitory activity and those of the complete reaction mixture as 0%
inhibitory activity. Curve fittings and calculations of the IC50 values were performed using the
program XLfit ver. 5 (ID Business Solutions Ltd.), with the maximum and minimum of the
curve constrained to 100 and 0, respectively.
May–Grünwald Giemsa staining
For a morphological analysis of human leukemia cell lines, we prepared cytospin slides via
centrifugation at 700 rpm for 3 min on Cytospin 4 positively-charged glass slides (Thermo
Fisher Scientific, San Jose, CA, USA). The slides were stained with May–Grünwald solution
(Wako, Osaka, Japan) in PBS (pH 6.4) for 3 min at room temperature and washed briefly.
Giemsa solution (Wako) was added to each slide, followed by a 15-min incubation. Cellular
morphology was evaluated on the stained slides using an Axiokop 2 plus microscope with
NanoZoomer Digital Pathology (Hamamatsu Photonics, Shizuoka, Japan).
Wash out assay
TF-1a cells were treated with 1 μM T-3775440 for 72 h and subsequently stained with
APC-conjugated anti-human CD86 antibody (305412; BioLegend, San Diego, CA, USA) in
FcR Blocking Reagent (130-059-901; Miltenyi Biotec, Auburn, CA, USA). Stained cells were
sorted into CD86 high, middle, or low populations on a FACSAria II™ (Becton-Dickinson,
Franklin Lakes, NJ, USA). Sorted cells were seeded in 96-well plates and incubated with
T-3775440-free growth media. After 3 and 5 days, growth ratios were measured using
CellTiter-Glo (Promega).
Surface marker array
TF-1a and HEL92.1.7 cells were respectively incubated with 50 nM and 100 nM T-3775440 for
72 h. Treated cells were stained with BD Lyoplate™ (560747; BD Biosciences, San Diego, CA,
USA) according to the manufacturer's instructions, and analyzed on an LSRFortessa™
(Becton-Dickinson). Mean fluorescent intensities (MFIs) were calculated according to the BD
3
Lyoplate™ Human Screen Analysis Instructions for BD FACSDiva, which are available as an
Excel file on the manufacturer's web site.
siRNA transfection
For each targeted gene, siRNA was obtained from Dharmacon (Chicago, IL, USA) or Ambion
(Austin, TX, USA) as described in the table below. siRNA was formulated into lipid-based
nanoparticles. Nanoparticulated siRNA was mixed with cells at a final concentration of 10 nM.
For drug treatment experiments, cells were reseeded into tissue culture plates at 48 h
post-siRNA transduction and treated with DMSO or drugs. The following siRNAs were used in
this study:
siRNA
ID
Format
Vendor
siCTRL
siKDM1A #1
siKDM1A #2
siGFI1B #1
siGFI1B #2
siSPI1 #1
siSPI1 #2
D-001810-10
L-009223-00
118783
s15850
s15851
s13351
s13352
ON-TARGETplus Non-targeting pool
SMARTpool: ON-TARGETplus KDM1A siRNA
Silencer Validated siRNA
Silencer Select Pre-Designed siRNA
Silencer Select Pre-Designed siRNA
Silencer Select Pre-Designed siRNA
Silencer Select Pre-Designed siRNA
Dharmacon
Dharmacon
Ambion
Ambion
Ambion
Ambion
Ambion
Surface plasmon resonance (SPR) experiment
SPR biosensing experiments were performed on a BiacoreS200 with a CM5 sensorchip (GE
Healthcare) at 25ºC. HBS-P+ (10 mM Hepes, pH 7.4, 150 mM NaCl, 0.005% SP20)
supplemented with 1 mM DTT was used as the running buffer for our immobilization and
interaction study. The LSD1/CoREST complex was immobilized on the sensorchip using a
standard amine coupling procedure, according to the manufacturer's instructions. The final
immobilization level was approximately 10000 RUs. The compounds and GFI1B peptide
(PRSFLVKSKKAHTYHQPPRVQ, purchased form SCRUM Inc.) were stored as 10 mM stock
solutions in 100% DMSO. These solutions were diluted 1:1000 (v/v) in running buffer. GFI1B
peptide at 1 µM was injected for 120 s at a flow rate of 50 µL/min, and dissociation was
followed thereafter for up to 240 s. To inactivate the LSD1/CoREST complex, T-3775440 at 10
µM was injected for 180 s at a flow rate of 50 µL/min. BIAevaluation ver. 4.1.1 (GE
Healthcare) was used for data processing after subtraction of the reference and blank samples.
Chromatin immunoprecipitation assay
TF-1a cells were treated with 100 nM of T-3775440 or DMSO for 24 h. A chromatin
immunoprecipitation assay was performed using a SimpleChIP Plus Enzymatic Chromatin IP
Kit (Cell Signaling Technology, Danvers, MA, USA). Antibodies against the following proteins
were used for immunoprecipitation or western blot analysis: normal rabbit IgG (12–370;
4
Millipore, Billerica, MA, USA), LSD1 (ab17721; Abcam, Cambridge, UK), GFI1B (5849; Cell
Signaling Technology), and dimethyl histone H3 Lys4 (07–030; Millipore). Precipitated DNA
was analyzed by quantitative real-time PCR. The following primers were used for PI16:
5'-ACTTTAAATCCAGCTGCCAGAC-3'
(forward)
and
5'-CAGCAGAAGCATCAGGAAACT-3' (reverse). For RPL3, the primers included with the
above-mentioned ChIP kit were used.
In vitro colony-forming cell assays
Three different lots of human bone marrow mononuclear cells were purchased from commercial
suppliers (two lots from Lonza [Walkersville, MD, USA]; one lot from Stemexpress [Placerville,
CA, USA]). Hematopoietic progenitor frequencies were quantified using a colony-forming cell
assay at STEMCELL Technologies Inc. (Vancouver, BC, Canada). Appropriate numbers of
qualified cells were incubated with T-3775440 or an equivalent amount of DMSO in
methylcellulose-based medium (MethoCult™ GF H84434, STEMCELL) containing G-CSF,
GM-CSF, IL-3, SCF, and EPO. After 14 days in culture, hematopoietic progenitor colonies
(CFU-E, BFU-E, CFU-GM, and CFU-GEMM) were enumerated. The mean colony number was
calculated for triplicate cultures at each condition. The percentage of growth control for each
progenitor type (erythroid and myeloid) was calculated at each T-3775440 test concentration,
and a dose-response curve was generated to calculate the IC50 values.
5
Supplementary Figures
Supplementary Figure S1. T-3775440 inhibits AML cell proliferation.
(A) Growth inhibition curves of representative leukemia cell lines treated with T-3775440. (B)
Erythroleukemia cell line HEL92.1.7 cells and megakaryoblastic leukemia cell line M07e cells
were treated with the indicated concentrations of T-3775440 for 24 or 48 h. Subsequently, cells
were collected, and total lysates were prepared. Immunoblotting analyses were performed to
determine the expression levels of p27, cleaved PARP, and β-actin in the cell lysates.
6
Supplementary Figure S2. T-3775440 modulates gene signatures in AML cell lines.
(A) HEL92.1.7 and CMK11-5 cells were treated with DMSO or T-3775440 for 24 h and
subjected to a microarray analysis. The Venn diagram illustrates the number of probes that were
commonly upregulated by treatment with T-3775440 (p < 0.01, ≥2-fold change). (B) Data were
generated as in (A), but the number of probes that were significantly decreased upon treatment
of T-3775440 is shown. (C, D) A gene set enrichment analysis (GSEA) plot shows the
upregulation of natural killer signature genes (C) and monocytic signature genes (D) in
CMK11-5 cells following treatment with T-3775440 versus a control. (E) A GSEA plot
illustrates downregulation of the megakaryocytic signature genes as in (C, D).
7
Supplementary Figure S3. Transcriptional and immunophenotypical changes after
treatment with T-3775440. (A) Induction of CD86 expression in HEL92.1.7 cells upon
treatment with 100 nM T-3775440 for 72 h, as measured by flow cytometry. (B) The reduction
in CD235a expression was monitored as in (A). (C) CMK11-5 cells were incubated in the
presence or absence of T-3775440 for 72 h. After treatment, the cell surface expression of CD86
protein was analyzed using flow cytometry. (D-F) Cells were treated with DMSO or T-3775440
for 24 h. CD86 gene expression changes were measured by qRT-PCR in CMK11-5 (D),
HEL92.1.7 (E), and TF-1a cells (F). Values represent the means of triplicate samples ± standard
deviations.
8
Supplementary Figure S4. SPI1 depletion reversed the transcriptional changes induced by
T-3775440. (A–D) SPI1 was knocked down by siRNA treatment in TF-1a cells, which were
subsequently reseeded in the presence or absence of 100 nM T-3775440. Twenty-four hours
after treatment with the compound or vehicle control, cells were harvested for RNA purification.
Changes in SPI1 (A), FCRLA (B), ITGAM (C), and GATA1 (D) expression were measured by
qRT-PCR. Values represent the means of triplicate samples ± standard deviations. Asterisks
denote a p < 0.01 (**), as determined by the Dunnett-type test.
9
Supplementary Figure S5. GSK-LSD1 inhibits the LSD1–GFI1B interaction. Surface
plasmon resonance (SPR) sensorgrams for the interaction between GFI1B peptide and LSD1 are
indicated in gray. The interaction between GFI1B peptide and LSD1 after treatment with 10 µM
of GSK-LSD1 is indicated in red.
10
Supplementary Figure S6. T-3775440 increases PI16 gene expression in vitro. (A, B) Time(A) and dose-dependent (B) effects on PI16 expression were observed in TF-1a cells in vitro.
Cells were treated with 100 nM T-3775440 and harvested at the indicated time points (A). (B)
HEL92.1.7 cells were incubated in the presence or absence of T-3775440 for 24 h. Changes in
PI16 expression were measured by qRT-PCR. Values represent the means of triplicate samples ±
standard deviations. (C, D) HEL92.1.7 cells were subjected to the experiments described in (A
and B). (E) TF-1a cells were treated with 100 nM T-3775440 or DMSO control for 24 h. After
treatment, chromatin was cross-linked and sonicated, and the samples were subjected to
chromatin immunoprecipitation for the indicated antibodies. ChIPed DNA was used as a
template for quantitative PCR to determine the relative enrichment on the RPL3 gene body.
Values represent the means of triplicate samples ± standard deviations (SDs).
11
Supplementary Figure S7. RNAi-mediated knockdown of LSD1 and GFI1B recapitulates
the gene expression changes observed with T-3775440 treatment. (A–F) TF-1a cells were
treated with the indicated siRNAs, and the mRNA expression levels of LSD1 (A), GFI1B (B),
FCRLA (C), ITGAM (D), PI16 (E), and GATA1 (F) were measured by qRT-PCR after a 48-h
treatment. Values represent the means of triplicate samples ± standard deviations.
12
Supplementary Figure S8. RNAi-mediated knockdown of LSD1 and GFI1B induces
morphological changes in AML cells. TF-1a cells were treated with the indicated siRNAs and
harvested after 72 h of treatment. Cells were stained with May–Grünwald Giemsa stain.
Representative images of cytospin preparations are shown. Cells were observed at 40×
magnification using a light microscope.
13
Supplementary Figure S9. Dose-response curves of the effects of T-3775440 on erythroid
and myeloid progenitor proliferation. The average colony counts per dish were obtained from
cultures of each bone marrow lot (A and D, BM856233002; B and E, BM070525B; C and F,
BM070973A). (A–C) Myeloid progenitors: CFU-GM. (D–F) Erythroid progenitors: CFU-E and
BFU-E. Values are expressed as percentages of the numbers of colonies in DMSO-treated
controls. IC50 values are also presented for b, d, and f.
14
Supplementary Figure S10. T-3775440 treatment causes a transient reduction in platelets
in mice, followed by a rebound. (A, B) Peripheral platelet (A) and red blood cell (RBC) (B)
counts in ICR mice were analyzed using a Sysmex XT-1800i Automated Hematology Analyzer
(Sysmex, Kobe, Japan) before and after a single oral administration of T-3775440. *P < 0.05,
***P < 0.001 by Dunnett's multiple comparison test.
15
Supplementary Tables
Supplementary Table S1. Culture medium information and IC50 values of T-3775440 from the
in vitro proliferation assay.
Cell line
FAB classification
Medium
FBS
Supplement
IC 5 0 (M)
Year
obtained
Kasumi-1 (JCRB)
AML (M2)
RPMI 1640 (wako, 189-02145)
10%
no
1.3E-08
2014
M07e (DSMZ)
AML (M7)
RPMI 1640 (wako, 189-02145)
20%
10ng/ml rhGM-CSF (wako, 075-04114)
1.8E-08
2014
CMK-86 (JCRB)
AML (M7)
RPMI 1640 (wako, 189-02145)
10%
no
2.0E-08
2014
OCI-M2 (DSMZ)
AML (M6)
IMDM; gibco, Cat No: 12440-053
20%
no
2.5E-08
2008
CMK-11-5
(JCRB)
AML (M7)
RPMI 1640 (wako, 189-02145)
10%
no
2.5E-08
2014
TF-1a (ATCC)
AML (M6)
RPMI 1640 (wako, 189-02145)
10%
no
3.9E-08
2014
EOL-1 (DSMZ)
AML (eosinophilic
leukemia)
RPMI 1640 (wako, 189-02145)
10%
no
7.4E-08
2014
HEL92.1.7(ATCC)
AML (M6)
RPMI 1640 (wako, 189-02145)
10%
no
4.9E-07
2014
TF-1 (ATCC)
AML (M6)
RPMI 1640 (wako, 189-02145)
10%
2ng/ml rhGM-CSF (wako, 75-04114)
2.0E-06
2014
Kasumi-3 (JCRB)
AML (M0)
RPMI 1640 (wako, 189-02145)
10%
no
3.3E-06
2014
ML-2 (DSMZ)
AML (M4)
RPMI 1640 (wako, 189-02145)
10%
no
>1.0E-005
2014
HL-60 (ATCC)
AML (M2)
IMDM (gibco, 12440-053)
20%
no
>1.0E-005
2003
KG-1 (ATCC)
AML (M6)
IMDM; gibco, Cat No: 12440-053
20%
no
>1.0E-005
2002
KU812F (ATCC)
CML
RPMI 1640 (wako, 189-02145)
10%
no
>1.0E-005
2014
SKM-1 (JCRB)
MDS/AML (M5)
RPMI 1640 (wako, 189-02145)
10%
no
>1.0E-005
2014
NB4 (DSMZ)
AML (M3)
RPMI 1640 (wako, 189-02145)
10%
no
>1.0E-005
2009
16
Supplementary Table S1 (Continued). Culture medium information and IC50 values of
T-3775440 from the in vitro proliferation assay.
Cell line
FAB classification
Medium
FBS
Supplement
IC 5 0 (M)
Year
obtained
OCI-AML3
(DSMZ)
AML (M4)
MEM α (wako, 135-15175)
20%
no
>1.0E-005
2014
MOLM-16
(DSMZ)
AML (M0)
RPMI 1640 (wako, 189-02145)
20%
no
>1.0E-005
2014
PL-21 (JCRB)
AML (M3)
RPMI 1640 (wako, 189-02145)
20%
no
>1.0E-005
2014
MOLT-4 (ATCC)
T-ALL
RPMI 1640 (wako, 189-02145)
10%
no
>1.0E-005
2014
DND-41 (DSMZ)
T-ALL
RPMI 1640 (wako, 189-02145)
10%
no
>1.0E-005
2014
TALL-1 (DSMZ)
T-ALL
RPMI 1640 (wako, 189-02145)
15%
no
>1.0E-005
2014
HPB-ALL (DSMZ)
T-ALL
RPMI 1640 (wako, 189-02145)
20%
no
>1.0E-005
2014
THP-1 (ATCC)
AML (M5)
RPMI 1640 (wako, 189-02145)
10%
0.05mM 3-mercapto-1,2-propandiol
(wako, 139-16452)
>1.0E-005
2010
NOMO-1 (JCRB)
AML (M5)
RPMI 1640 (wako, 189-02145)
10%
no
>1.0E-005
2014
MKPL-1 (JCRB)
AML (M7)
RPMI 1640 (wako, 189-02145)
20%
no
>1.0E-005
2014
GF-D8 (DSMZ)
AML (M1)
RPMI 1640 (wako, 189-02145)
20%
50ng/ml rhGM-CSF (wako, 075-04114)
>1.0E-005
2008
17
Supplementary Table S2. List of enriched pathways from commonly upregulated genes in both
HEL92.1.7 and CMK11-5 cells by the treatment of T-3775440.
B iogroup
S ourc e
Immunoglobulin-like fold
InterPro
6.70E-11
Immunoglobulin-like
InterPro
7.40E-11
cell periphery
GO
1.50E-09
immune response
GO
2.00E-08
SPI1 binding site geneset 2
Broad MSigDB - Regulatory Motifs
6.90E-08
single-organism process
GO
1.00E-07
ETS2 binding site geneset 2
Broad MSigDB - Regulatory Motifs
1.20E-07
Immunoglobulin subtype
InterPro
1.70E-07
response to stress
GO
9.30E-07
Immunoglobulin V-set
InterPro
1.90E-06
cellular response to stimulus
GO
2.40E-06
SPI1 binding site geneset 1
Broad MSigDB - Regulatory Motifs
2.60E-06
SOX9 binding site geneset 1
Broad MSigDB - Regulatory Motifs
3.10E-06
single organism signaling
GO
3.90E-06
leukocyte migration
GO
4.10E-06
response to bacterium
GO
4.90E-06
ELF1 binding site geneset 1
Broad MSigDB - Regulatory Motifs
4.90E-06
ETV4 binding site geneset 1
Broad MSigDB - Regulatory Motifs
8.80E-06
response to other organism
GO
1.80E-05
FOXD1 binding site geneset 1
Broad MSigDB - Regulatory Motifs
2.70E-05
regulation of immune response
GO
2.70E-05
response to wounding
GO
2.80E-05
HMGA1 binding site geneset 1
Broad MSigDB - Regulatory Motifs
2.90E-05
NFAT NFATC binding site geneset 1
Broad MSigDB - Regulatory Motifs
3.60E-05
FOXF2 binding site geneset 3
Broad MSigDB - Regulatory Motifs
4.20E-05
Immunoglobulin Protein Family
InterPro
4.40E-05
MYOD1 binding site geneset 4
Broad MSigDB - Regulatory Motifs
5.20E-05
Beta-microseminoprotein
InterPro
6.20E-05
signaling receptor activity
GO
9.80E-05
18
p-v alue
Supplementary Table S3. List of surface molecules upregulated by T-3775440 treatment.
HE L92. 1. 7
TF-1a
A nt igen
DMS O
T-3775440
Rat io
DMS O
T-3775440
Rat io
CD86
2.75
29.45
10.71
2.01
13.68
6.80
CD11b
2.07
6.76
3.27
1.19
2.73
2.30
CD48
4.16
9.45
2.27
7.23
54.53
7.55
CD39
2.98
6.46
2.17
2.51
7.79
3.10
CD201
20.57
37.14
1.81
3.85
24.25
6.29
CD99R
11.02
19.36
1.76
1.68
3.66
2.18
Data are presented as mean fluorescence intensity values.
19
Supplementary Table S4. List of surface molecules downregulated by T-3775440 treatment.
HE L92. 1. 7
TF-1a
A nt igen
DMS O
T-3775440
Rat io
DMS O
T-3775440
Rat io
CD69
17.33
1.77
0.10
15.61
3.64
0.23
CD243
36.67
4.58
0.13
12.12
5.14
0.42
CD7
9.61
1.33
0.14
9.37
4.63
0.49
CD235a
375.27
54.74
0.15
139.74
61.68
0.44
CD36
95.23
15.67
0.16
36.71
6.41
0.17
CD206
3.88
0.71
0.18
2.14
1.03
0.48
CD274
6.01
1.17
0.19
14.78
6.04
0.41
CD71
100.52
19.97
0.20
29.36
10.01
0.34
CD220
42.72
8.93
0.21
3.13
1.02
0.33
CD227
29.00
6.80
0.23
13.80
5.16
0.37
CD29
4.51
1.21
0.27
5.32
1.50
0.28
CD62E
5.74
1.64
0.29
3.94
1.97
0.50
CD34
98.26
28.85
0.29
5.07
2.06
0.41
CD124
3.70
1.11
0.30
4.62
1.60
0.35
CD180
2.22
0.68
0.31
2.23
0.62
0.28
CD107a
10.44
3.32
0.32
7.73
3.35
0.43
CD226
180.94
62.12
0.34
30.19
12.96
0.43
CLIP
3.15
1.09
0.35
3.26
0.74
0.23
CD178
2.13
0.75
0.35
1.95
0.64
0.33
CD66f
2.08
0.74
0.36
2.57
0.80
0.31
CD102
132.44
48.55
0.37
47.39
20.54
0.43
CD164
51.27
20.00
0.39
22.50
7.97
0.35
CD27
2.25
0.90
0.40
2.72
1.36
0.50
CD49c
8.33
3.37
0.40
3.40
1.65
0.48
CD28
2.14
0.88
0.41
3.48
1.42
0.41
CD42a
2.06
0.90
0.44
6.87
1.36
0.20
CD121b
3.06
1.38
0.45
3.87
1.56
0.40
CD88
3.89
1.77
0.45
4.11
1.53
0.37
CD1d
10.99
5.10
0.46
14.33
7.11
0.50
Data are presented as mean fluorescence intensity values.
20