Figure legends
Figure 1 Effects of AZM on RA FLSs. (A) Following exposure to
TNF-α and IL-1β for 6 h, RA FLSs (n=3) were treated by AZM or Vehicle
for another 24 h. IL-6 (A) , IL-8 (B) , MMP-1(C) and MMP-3 (D) levels in the supernatant of RA FLSs
were analysed by ELISA. The migration (E) and invasion(F) abilities were assessed in AZM or Vehicle treated RA FLSs
from six different patients (3 males and 3 females). Data represent
independent experiments performed in triplicate, and six different
fields were selected for cell counting (graph below). RA FLSs passing
through the polycarbonate membrane with ECM coating shows that cell
invasion requires the ECM proteolysis step in addition to migration. RA
FLSs (n=3) were treated by AZM or Vehicle for 24 h, followed by TNF-α or
IL-1β stimulation for 6 h. CXCL9 (G) , CXCL10 (H)levels in the culture supernatant were measured by ELISA. (I)After treatment as indicated in (A-D), cell-free RA FLSs supernatant was
collected and used as a chemo-tactic source for healthy donor peripheral
blood leucocytes (n=3) in a transwell migration system for 6 h. The
number of migrating leucocytes in the lower compartment was counted.
*P <0.05, **P <0.01 and
***P <0.001 compared with Vehicle. (J) The
production of VEGF decreased when RA FLSs were exposed to AZM.(K) Tube formation assay was applied to determine the
angiogenic ability of HUVECs after treatment with conditioned medium of
AZM or its Vehicle treated RA FLSs. Data in A-D, G and H are expressed
as the mean of three samples ± SD and represent three independent
experiments. *P <0.05, **P <0.01 and
***P <0.001 compared with Control. IL-1β,
interleukin-1β; TNF-α, tumour necrosis factor-α; AZM, azithromycin;
Ctrl, control.
Figure 2 Anti-arthritis effects of AZM on CIA. Mice immunized
with CII were randomly divided into 4 groups (n=6 mice for each group
and time point) and administered AZM or Vehicle at the indicated doses
twice a day after the initial immunization. The data are representative
of four independent experiments with similar results. (A)Arthritis scores were monitored once per five days. (B) Hind
paw thickness was calibrated from the 21st day following the first
immunization. AZM (low): #P <0.05,##P <0.01,###P <0.001 compared with Vehicle.
AZM (medium): *P <0.05, **P <0.01,
***P <0.001 compared with Vehicle. AZM (high):&P <0.05,&&P <0.01,&&&P <0.001 compared with Vehicle.(C) Fore paw (upper) and hind paw (lower) photographs obtained
on day 42 from mice with CIA from the day of first immunization.(D) Representative histology images by H&E staining about
interphalangeal joint (upper) and ankle joint (lower) obtained on day 67
from mice with CIA with the indicated AZM treatment. Pathological
changes, including synovial proliferation (red arrowhead) and joint
destruction (yellow arrowhead), are shown. (E) Inflammation,
hyperplasia, cartilage degradation and bone destruction were measured
through a scoring system (n= 12 mice per group). (F)Representative micro-CT images of hind paws and interphalangeal joints
(red square). (G) BV/TV, Tb. BMD, Tb. Th, Tb. N and Tb. Sp in
the distal tibia were assayed by micro-CT and 3D reconstruction. E and
G: ns: not significant, *P <0.05,
**P <0.01 and ***P <0.001 compared with
Vehicle group. CIA, collagen-induced arthritis; BV/TV, Bone volume
fraction; Tb. BMD, trabecular bone mineral density; Tb. Th, trabecular
thickness; Tb. N, trabecular number; Tb. Sp, trabecular separation.
Figure 3 Functional enrichment analysis of differentially
expressed genes. (A) Hierarchical clustering of the dysregulated mRNAs
in RA FLSs. The expression values were represented by a color scale. The
intensity increased from blue (relatively lower expression) to red
(relatively higher expression). Each column represented one tissue
sample, and each row represented a single mRNA. (B) GO analyses
of the host genes of differentially expressed mRNAs and GO annotations
of the host genes of differentially expressed mRNAs. The bar plot
presented the enrichment scores (−loge[p value]) of the top 10
significantly enriched GO terms in biological processes, cellular
components and molecular functions. (C) KEGG analyses of the
host genes of differentially expressed mRNAs and KEGG annotations of the
host genes of differentially expressed mRNAs. The bubble diagram
presented the enrichment scores (−loge[p value]) of the top 10
significantly enriched KEGG terms in molecular function and signal
pathway. (D) After stimulation with AZM for 24 h, total RNA was
extracted from RA FLSs and subjected to qRT-PCR for the assessment of
CHOP mRNA levels. qRT-PCR data are expressed as the mean ± SD of six
samples (3 males and 3 females) from two independent experiments. NS:
not significant, *P <0.05 and
***P <0.001 compared with vehicle. (E) Using
similar treatments as (D) , total and phosphorylated levels of
PERK and elF2α, and the increased expression of IRE1α, ATF4 were
analysed by western blot. Western blot data are representative of two
independent experiments from six different patients (3 male and 3
female) with similar results. (F) Under the stimulation of
TNF-α and IL-1β, similar treatment as (D) was used, total and
phosphorylated levels of p38 and Akt, and the increased expression of
CREB3L2 were analysed by western blot. Western blot data are
representative of two independent experiments from six different
patients (3 male and 3 female) with similar results. (G) RA
FLSs (n = 3) were treated with AZM or its vehicle for 24 h, Annexin X
staining were applied to detect the cell apoptosis of RA FLSs (n = 3).
The statistical significance of differences between AZM and
vehicle-treated groups was determined. Data represent the mean ± SD of
three independent replicates of three samples. **P <0.01
and ***P <0.001 compared with Control. DMSO, Dimethyl
Sulfoxide; Tm, tunicamycin; Tg, thapsigargin.
Figure 4 GRP78 is a novel target of AZM. (A) Coomassie blue
staining of DARTS assay. The band with molecular weight around 80 kDa
protected by AZM was indicated by arrow. (B) DARTS and Western
blot to confirm AZM’s binding targets. (C and D) CETSA melt
response. (E) DARTS assay with serial deletion constructs
encoding Flag-tagged mutants of GRP78. GRP78 (aa 1–750), GRP78 (aa
126–750), GRP78 (aa 406–750) and GRP78 (aa 1–479). 293T cells were
transfected with Flag tagged mutants of GRP78 plasmids, as indicated.
DARTS assay samples were detected by Flag antibody. (F)Potential targeting sites of AZM towards GRP78. (G) DARTS assay
for Asp-178 point mutant of cPLA2. The Asp-178 of cPLA2 was substituted
with Ala505. 293T cells were transfected with the point mutant plasmid
and DARTS was performed. Point mutated cPLA2 was detected by Flag
antibody. (H) The double reciprocal diagram of ATP
concentration and luminescence intensity under different concentrations
of AZM showed that the treatment of AZM might be related to ATP.(I) The enzyme activity of GRP78 was measured in the presence
of AZM or HA15. The initial concentration of Pronase was 10 mg/kg. ns:
not significant, **P <0.01 and
***P <0.001 compared with Vehicle. Temp, Temperature.
Figure 5 AZM inhibits GRP78 activity through binding to its
catalytic domain. (A) RA FLSs were stimulated with AZM for 24 h,
immunoprecipitation and immunoblotting were performed on total protein
with anti-GRP78 or anti- PERK, IRE1α or ATF6α antibodies. (B)Equal aliquots of nuclear and membrane protein from AZM or its vehicle
treated RA FLSs with GRP78 overexpression or not were pooled (total, 30
mg) and subjected to SDS-PAGE and immunoblot analysis for the indicated
protein. A and B: The results are representative of four samples (2 male
and 2 female) from independent experiment.
Figure 6 AZM activates UPR and promote apoptosis via GRP78. (A)GRP78 expression in Hela cells when GRP78 was knocked out using
CRISPR-Cas9 technique. (B) Total and phosphorylated levels of
PERK, elF2α and IRE1α, the expression of ATF6α (p50), ATF4, CHOP, as
well as the expression of m-SERBP-1c in membrane or nuclear
compartments, were detected in RA FLSs with GRP78 deficiency or not.
β-Actin was used as a loading control. (C) Silencing efficiency
of siRNA targeting GRP78 (siGRP78) was detected by western blot. The two
siRNAs were combined at equal concentrations for the subsequent
experiments. The negative control siRNA is referred to as siCtrl. The
results are representative of two independent experiments with three
different samples in each. (D) Following exposure to TNF-α and
IL-1β for 6 h, RA FLSs with GRP78 knocking down or not were treated by
AZM or Vehicle for another 24 h. IL-6, IL-8, MMP-1, MMP-3, CXCL9 and
CXCL10 levels in the supernatant of RA FLSs were analysed by ELISA. The
data are expressed as the mean ± SD of three samples and are
representative of three independent experiments.
***P <0.001 compared with SiCtrl. (E) RA FLSs
with GRP78 silencing or not were treated with AZM or its vehicle for 24
h, Annexin X staining were applied to detect the cell apoptosis of RA
FLSs (n = 3). ***P <0.001 compared with SiCtrl.
GRP78wt, GRP78 wild type. GRP78-/-,
GRP78 deficiency. GRP78mt, GRP78 mutation type.
Supplementary Figure 1 Effects of Etanercept and AZM on the
production of pro-inflammatory factors. Following exposure to TNF-α and
IL-1β for 6 h, RA FLSs were treated by AZM, Etanercept or in combinaton
for another 24 h. IL-6 (A) , IL-8 (B) , MMP-1(C) and MMP-3 (D) levels in the supernatant of RA FLSs
(n=3) were analysed by ELISA. Data are expressed as the mean ± SD of
three samples and are representative of three independent experiments.
ns: not significant, *P <0.05, **P <0.01
and ***P <0.001 compared with Vehicle group. ETC,
etanercept.
Supplementary Figure 2 Effects of AZM on the production of
pro-inflammatory factors in PBMC. Following exposure to TNF-α and IL-1β
for 6 h, RA FLSs were treated by AZM, Etanercept or in combination for
another 24 h. IL-6 (A) , IL-8 (B) , TNF-α (C) ,
IL-1α (D) and IL-1β (E) levels in the supernatant of
RA FLSs were analysed by ELISA. Data are expressed as the mean ± SD of
six samples (3 male and 3 female) and are representative of three
independent experiments. ns: not significant, *P <0.05,
**P <0.01 and ***P <0.001 compared with
Vehicle group.
Supplementary Figure 3 Effects of AZM on cytokine production in
CIA models. The serum from AZM or Vehicle treated CIA mice (n=6 mice
per group) were collected for ELISA detection of IL-1β and IL-6, COMP,
IL-10, IL-13, OPG and RANKL on the 42nd day after the first
immunization. This experiment data is representative of two independent
experiments from 6 mice per group. **P <0.01 and
***P <0.001 compared with Vehicle in the Control or CIA
group.
Supplementary Figure 4 qRT-PCR validation of fourteen
differentially expressed mRNAs in 20 pairs of RA FLSs samples. The
relative expression levels of 11 up-regulated and 3 down-regulated mRNAs
(selected from the top 10 dysregulated mRNAs) in 20 pairs of AZM and
Vehicle treated RA FLSs. ns: not significant, *P <0.05,
**P <0.01 and ***P <0.001 compared with
vehicle.
Supplementary Table 1 Clinical characteristics of RA patients.
Supplementary Table 2 Primer sequences of genes in real-time
fluorescent quantitative PCR (qRT-PCR).
Supplementary Table 3 List of potential AZM targets in RA FLSs
identified by mass spectrometry.
Supplementary
Table 4 Primer sequences of sgGRP78 in CRISPR/Cas9.