Figure legends
FIGURE 1 Inactivation of SERCA2 C674 suppresses PPARγ by
activation of calcineurin/NFAT/NF-κB pathways. (a) The main signaling
pathways from the Kyoto
encyclopedia of genes and genomes pathway enrichment analysis in
duplicate aorta samples of WT and SKI mice in LDLR−/−background. (b) The mRNA levels of PPARγ2 in aortas of WT and SKI mice
in LDLR−/− background. (c) Representative Western
blots of PPARγ from the aorta of WT and SKI mice in
LDLR−/− background, and quantification of band
intensities in graph. (d) The mouse PPARγ2 promoter activity in aortic
SMCs. (e) Effects of CsA or PDTC on PPARγ2 promoter activity in SKI
aortic SMCs. (f) Representative Western blots of PPARγ from aortic SMCs
and the quantification of band intensities in graph. (b)-(f), mean ±
SEM, unpaired Student’s t test, *P < .05,
significantly different as indicated, n = 5
FIGURE 2 Activation of NF-κB accounts for the phenotypic
modulation of SKI aortic SMCs. (a) Representative Western blots from SKI
aortic SMCs treated with PDTC and quantification of band intensities in
graph. Mean ± SEM, unpaired Student’s t test, *P< .05, significantly different as indicated, n = 5. (b)
Cell proliferation. (c) Cell migration. (d) Macrophage adhesion to
aortic SMCs. (b)-(d), mean ± SEM, unpaired Student’s t test,
*P < .05, significantly different as indicated,n = 6
FIGURE 3 The down-regulated PPARγ2 accounts for the phenotypic
modulation of SKI SMCs. (a) Representative Western blots from SKI aortic
SMCs transfected with PPARγ2 plasmids and quantification of band
intensities in graph. Mean ± SEM, unpaired Student’s t test,
*P < .05, significantly different as indicated,n = 5. (b) Cell proliferation. (c) Cell migration. (d) Macrophage
adhesion to aortic SMCs. (b)-(d), mean ± SEM, unpaired Student’st test, *P < .05, significantly different as
indicated, n = 6
FIGURE 4 Activation of PPARγ by pioglitazone prevents the
phenotypic modulation of SKI SMCs. (a) Representative Western blots from
SKI aortic SMCs treated with pioglitazone and quantification of band
intensities in graph. Ctrl, solvent control; PIOG, pioglitazone. Mean ±
SEM, unpaired Student’s t test, *P < .05,
significantly different as indicated, n = 5. (b) Cell
proliferation. (c) Cell migration. (d) Macrophage adhesion to aortic
SMCs. (b)-(d), mean ± SEM, unpaired Student’s t test, *P< .05, significantly different as indicated, n = 6
FIGURE 5 Activation of PPARγ represses the activation of
NFAT/NF-κB in SKI aortic SMCs. Representative Western blots of NFAT4 and
p65NF-κB from SKI aortic SMCs treated with pioglitazone and
quantification of band intensities in graph. Histone H3 is the load
control of nuclear protein and GAPDH is the load control of total
protein. Ctrl, solvent control; PIOG, pioglitazone. Mean ± SEM, unpaired
Student’s t test, *P < .05, significantly
different as indicated, n = 5
FIGURE 6 Activation of PPARγ ameliorates angiotensin II-induced
aortic aneurysm in SKI mice. (a) The flow chart of animal procedure in
LDLR−/− background mice. (b) Survival rate. SKI mice
treated with solvent control (SKI/Ctrl) vs. WT mice treated with solvent
control (WT/Ctrl) or SKI mice treated with pioglitazone (SKI/PIOG),
*P < .05, Kaplan-Meier analysis and comparisons are
performed using the log-rank test. (c) Incidence. (d) Representative
photographs of aortic aneurysms and the summary of their composition in
graph. A red arrow indicates the location of the aneurysm. Ruptured
aneurysm, SKI/Ctrl vs. WT/Ctrl or SKI/PIOG, *P < .05,
Chi-square test. (b)-(d), n = 11. (e) Representative photographs
of elastin degradation and the percentage of different grades of elastin
degradation summarized in graph.
Grade IV, SKI/Ctrl vs. WT/Ctrl or
SKI/PIOG, *P < .05, Chi-square test. (f) Representative
photographs of collagen deposition and its score summary in graph.
(e)-(f), scale = 500 μm. Mean ± SEM, WT/Ctrl, n = 8; SKI/Ctrl,n = 7; SKI/PIOG, n = 9
FIGURE 7 Activation of PPARγ decreased the size and number of
angiotensin II-induced aortic aneurysm in SKI mice. (a) Representative
photographs of HE staining of aortic aneurysms and the diameter and area
of aneurysm summarized in graph. Scale = 500 μm. (b) The length of
aneurysm. (c) The average number of aortic aneurysms in each group. Mean
± SEM, One-way ANOVA, *P < .05, significantly different
as indicated, WT/Ctrl, n = 8; SKI/Ctrl, n = 7; SKI/PIOG,n = 9
FIGURE 8 Pioglitazone therapy inhibited the expression of
aneurysm related proteins in angiotensin II-infused SKI aorta. (a)
Representative Western blots from the aorta of SKI mice in
LDLR−/− background infused with angiotensin II, and
quantification of band intensities in graph. Ang II, angiotensin II;
Ctrl, solvent control; PIOG, pioglitazone. Mean ± SEM, unpaired
Student’s t test, *P < .05, significantly
different as indicated, n = 5