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Figure legends
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Figure 1. Overview of experimental design for biomarker discovery
Serum EVs were purified by size exclusion chromatography using sera from 10 patients
with PM/DM, 18 with RA, 5 SLE patients, and 10 HCs as the screening set (n=43).
LC/MS was applied to identify all proteins contained in the serum EVs. Several
biomarker candidates were selected by bioinformatics and statistical analyses. Plexin D1
was identified as a novel biomarker candidate by verification using Western blotting.
Further, we established a specific EV sandwich ELISA for detecting Plexin D1-positive
EVs and evaluated the clinical utility of such EVs using sera from 54 PM/DM, 24 RA,
20 SLE, 13 SSc, 25 DMD/BMD patients, and 36 HCs as the validation set (n=172).
DMD/BMD: Duchenne and Becker muscular dystrophy; EV: extracellular vesicle; HC:
healthy control; LC/MS: liquid chromatography/mass spectrometry; PM/DM:
polymyositis and dermatomyositis
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Figure 2. Plexin D1 in serum extracellular vesicles is a novel candidate biomarker of
polymyositis and dermatomyositis
(A) Volcano plot showing up- or down-regulated proteins in serum EVs of PM/DM
patients relative to HCs (left) or other patients (right). The x-axis indicates the log2 (foldchange) and the y-axis indicates the -log10 (P-value). Dashed lines indicate the thresholds,
p<0.05 (unpaired Student’s t-test) and fold change ≥2.0 or ≤0.5. The up- or downregulated proteins were assessed for their membrane or non-membrane nature using
DAVID gene ontology and UniProtKB/Swiss-plot. Black or white circles represent the
up-regulated membrane or non-membrane proteins in serum EVs of PM/DM patients,
respectively. White squares represent the down-regulated non-membrane proteins in
serum EVs of PM/DM patients. CYBB, ICAM5, NEO1, PLXD1, and SLC1A2 are
common up-regulated membrane proteins in serum EVs of PM/DM patients compared to
HC and RA and SLE patients. (B) Plexin D1 levels in serum EVs of 10 HC, 10 PM/DM
and 23 other patients were quantified by LC/MS analysis. The horizontal line represents
the median value. The P-value was calculated using the Kruskal-Wallis test with Dunn's
post hoc testing (multiple comparisons versus PM/DM patients). (C, D) Verification of
Plexin D1 in serum EVs. The pooled serum of PM/DM patients was fractionated by size
exclusion chromatography. The expression of Plexin D1 in serum EVs-containing eluates
was confirmed by Western blotting (C) and EV sandwich ELISA for detecting CD9- and
Plexin D1-double-positive EVs (CD9+ Plexin D1+ EVs) (D). Serum EVs-containing
fractions were determined by EV sandwich ELISA detecting for CD9- and CD63-double16
positive EVs (CD9+ CD63+ EVs) and protein concentration by micro-BCA protein assay.
EV: extracellular vesicle; HC: healthy control; LC/MS: liquid chromatography/mass
spectrometry; PM/DM: polymyositis and dermatomyositis
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Figure 3. Serum levels of Plexin D1-positive extracellular vesicles are higher in PM/DM
patients with associated muscle pain or weakness
Using 172 serum samples, serum levels of Plexin D1-positive EVs were measured by a
specific EV sandwich ELISA for directly detecting serum CD9- and Plexin D1-doublepositive EVs (CD9+ Plexin D1+ EVs). The index was calculated by optical density from
serum samples and standard control sample. (A) 54 PM/DM, 24 RA, 20 SLE, 13 SSc and
36 HCs. (B) 54 PM/DM, 25 DMD/BMD, and 36 HC. (C) Clinical subtype of PM/DM
patients (14 PM, 20 classic DM, 20 clinically amyopathic dermatomyositis (CADM)).
(D) myositis-specific autoantibody (MSA)-associated subtype of PM/DM patients with
anti-aminoacyl-tRNA synthetase (ARS) antibody (n=17), anti-melanoma differentiationassociated gene 5 (MDA5) antibody (n=16) and without either antibody (Others, n=22).
Associations between serum Plexin D1-positive EVs and (E) ILD, (F) muscle pain or
weakness, (G) dysphagia, and (H) malignancy in PM/DM patients. The horizontal line
represents the median value. The P-value was calculated using the Kruskal-Wallis test
with Dunn's post hoc testing (multiple comparisons versus PM/DM patients (A, B) or HC
(C, D)) and Wilcoxon rank-sum testing (E-H).
DMD/BMD: Duchenne and Becker muscular dystrophy; EV: extracellular vesicle; HC:
healthy control; ILD: interstitial lung disease; LC/MS: liquid chromatography/mass
spectrometry; PM/DM: polymyositis and dermatomyositis
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Figure 4. Serum levels of Plexin D1-positive extracellular vesicles correlate with
amounts of aldolase, white blood cells, neutrophils, and platelets in PM/DM patients
Correlations between serum levels of CD9+ Plexin D1+ EVs and (A) C-reactive protein
(CRP), (B) creatine kinase (CK), (C) aldolase, (D) Krebs von den Lungen-6 (KL-6), (E)
ferritin, (F) white blood cells (WBC), (G) neutrophils, (H) lymphocytes, (I) monocytes,
(J) platelets (PLT) in pre-treatment PM/DM patients. The black line shows the regression
line. The Spearman rank correlation coefficients (rs) and corresponding P-values are
indicated on each scatter plot.
EV: extracellular vesicle; PM/DM: polymyositis and dermatomyositis
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Figure 5. Serum levels of Plexin D1-positive extracellular vesicles are decreased in
PM/DM patients after treatment.
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Change of serum levels of CD9+ Plexin D1+ EVs in PM/DM patients after treatment in
21 PM/DM (9 PM, 7 DM and 5 clinically amyopathic dermatomyositis (CADM)) patients
who achieved clinical remission (A) and 3 PM/DM (3 DM) patients who did not (B).
Black circles connected by a solid line represent data from one individual patient. The Pvalue was calculated using the Wilcoxon signed-rank test.
EV: extracellular vesicle; PM/DM: polymyositis and dermatomyositis
18
Table 1 Characteristics of PM/DM patients and control group
Screening set (n=43)
Validation set (n=172)
Control group
Control group
PM/DM
RA
SLE
HC
PM/DM
RA
SLE
SSc
DMD/BMD
HC
Number of samples
10
18
10
54
24
20
13
25
36
Age (years), mean (SD)
44.5 (18.6)
62.9 (12.6)
31.2 (10.8)
50.8 (6.0)
54.1 (15.7)
57.3 (11.3)
38.1 (16.9)
60.1 (9.2)
14.4 (6.4)
48.9 (7.2)
Female, n (%)
7 (70.0)
15 (83.3)
4 (80.0)
7 (70.0)
38 (70.4)
16 (66.7)
16 (80.0)
8 (61.5)
1 (4.0)
24 (66.7)
Subtype of PM/DM
PM, n (%)
2 (20.0)
14 (26.0)
Classic DM, n (%)
3 (30.0)
20 (37.0)
CADM, n (%)
5 (50.0)
20 (37.0)
Anti-ARS, n (%)
2 (20.0)
17 (31.5)
Anti-MDA5, n (%)
5 (50.0)
16 (29.6)
Rash typical of DM, n (%)
8 (80.0)
40 (74.1)
ILD, n (%)
8 (80.0)
40 (74.1)
Muscle pain or weakness, n (%)
6 (60.0)
33 (61.1)
Dysphagia, n (%)
2 (20.0)
14 (25.9)
Malignancy, n (%)
0 (0)
6 (11.1)
MSA
Clinical symptoms
PM/DM: polymyositis/dermatomyositis; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; SSc: systemic sclerosis; DMD/BMD: Duchenne and Becker muscular
dystrophy; HC: healthy controls; CADM: clinically amyopathic dermatomyositis; MSA: myositis-specific autoantibody; ARS: aminoacyl-tRNA synthetase;
MDA5: melanoma differentiation-associated gene 5; ILD: interstitial lung disease.
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