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Supplementary Table S1. List of miRNA mimics and siRNAs used for transfection
miRNA
Catalog number
Source
mmu-miR-582-5p miRCURY LNA miRNA mimic
YM00471571
Qiagen, Hilden, Germany
control miRCURY LNA miRNA mimic
YM00479902
Qiagen, Hilden, Germany
Skp1-siRNA
MSS277837
Invitrogen, Carlsbad, CA, USA
control siRNA
S10C-0600
Cosmo Bio, Tokyo, Japan
siRNA
Supplementary Table S2. Sequence of primers used in this study
Gene name
Gapdh
Myd88
Irak4
Traf6
Tak1
Tab1
Tab2
Skp1
Tnfa
Il-1b
Il-6
Sequence
forward
5′-AATGTGTCCGTCGTGGATCTGA-3′
reverse
5′-GATGCCTGCTTCACCACCTTCT-3′
forward
5′-AGGACAAACGCCGGAACTTTT-3′
reverse
5′-GCCGATAGTCTGTCTGTTCTAGT-3′
forward
5′-CCTGGATGTCCTGGAACTTG-3′
reverse
5′-CAACACGCAGTAGGCAGAGA-3′
forward
5′-ACTGGGGACAATTCACTAGAGC-3′
reverse
5′-AAAGCGAGAGATTCTTTCCCTG-3′
forward
5′-AGGTTGTCGGAAGAGGAGCT-3′
reverse
5′-CTCCACAATGAAAGCCTTCC-3′
forward
5′-ACCCTGCTGGTGAGGAACT-3′
reverse
5′-AGGGACAGAGTCACACTAGTCT-3′
forward
5′-GGATAGAATAAGCGAAGCCCGGAA-3′
reverse
5′-CTCTTTGAAGCCGTTCCATCCT-3′
forward
5′-ATGCCTACGATAAAGTTGCAGA-3′
reverse
5′-TCCATTCCCAAATCTTCCAGC-3′
forward
5′-CATGGATCTCAAAGACAACC-3′
reverse
5′-GGTATATGGGCTCATACCAG-3′
forward
5′-GAAGAAGAGCCCATCCTCTG-3′
reverse
5′-TCATCTCGGAGCCTGTAGTG-3′
forward
5′-TGCCTTCTTGGGACTGATG-3′
reverse
5′-ACTCTGGCTTTGTCTTTCTTGT-3′
RNU6-6P
5′-CGCAAGGATGACACGCAAATTCGT-3′
mmu-miR-582-5p
5′-GGTATATGGGCTCATACCAG-3′
Gapdh: glyceraldehyde-3-phosphate dehydrogenase; Myd88: myeloid differentiation primary
response gene 88; Irak4: interleukin-1 receptor-associated kinase 4; Traf6: TNF receptorassociated factor 6; Tak1: mitogen-activated protein kinase kinase kinase 7; Tab1: TGF-beta
activated kinase 1/MAP3K7 binding protein 1; Tab2: TGF-beta activated kinase 1/MAP3K7
binding protein 2; Skp1: S-phase kinase-associated protein 1; Tnfa: tumor necrosis factor-alpha;
Il-1b: interleukin-1b; Il-6: interleukin-6; RNU6-6P: U6 small nuclear 6
Supplementary Table S3. List of antibodies used in western blotting and immunofluorescence staining
Antibody (Catalog number)
Source
TNF-α (AB-401-NA)
R&D Systems, Minneapolis, MN, USA
IL-1β /IL-1F2 (AF-401-NA)
R&D Systems, Minneapolis, MN, USA
IL-6 (AB-406-NA)
R&D Systems, Minneapolis, MN, USA
SKP1 (H-6; sc-5281)
Santa Cruz Biotechnology, Dallas, TX, USA
SKP1 (GTX106675)
GeneTex, Irvine, CA, USA
IκBα antibody (#9242)
Cell Signaling Technology, Beverly, MA, USA
phospho- NF-κB p65 (Ser536; #3033)
Cell Signaling Technology, Beverly, MA, USA
NF-κB p65 (D14E12; #8242)
Cell Signaling Technology, Beverly, MA, USA
β-actin (#4967)
Cell Signaling Technology, Beverly, MA, USA
Histone H1 (AE-4; sc-8030)
Santa Cruz Biotechnology, Dallas, TX, USA
LaminB1 (12987-1-AP)
ProteinTech, Rosemont, IL, USA
βTrCP (sc-390629)
Santa Cruz Biotechnology, Dallas, TX, USA
CUL-1 (sc-17775)
Santa Cruz Biotechnology, Dallas, TX, USA
RBX1 (sc-393640)
Santa Cruz Biotechnology, Dallas, TX, USA
HRP-conjugated anti-rabbit IgG (#7074)
Cell Signaling Technology, Beverly, MA, USA
HRP-conjugated anti-mouse IgG (#7076)
Cell Signaling Technology, Beverly, MA, USA
HRP-conjugated Rabbit Anti-Goat IgG (SA00001-4)
ProteinTech, Rosemont, IL, USA
Alexa Fluor 488-conjugated goat anti-rabbit IgG (A31627)
Invitrogen, Carlsbad, CA, USA
Alexa Fluor 488-conjugated goat anti-mouse IgG (A28175)
Invitrogen, Carlsbad, CA, USA
TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1 beta; IL-6: interleukin-6; SKP1: S-phase kinaseassociated protein 1; IκBα: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha;
NF-κB: nuclear factor-kappa B; βTrCP: beta transducin repeat containing protein; CUL-1: cullin 1; RBX1:
ring-box 1
Supplementary Fig. S1
Food intake
g per day
Body weight
(g)
60
40
20
ND
ND
HFD
HFD
ND
HFD
10 11 12 13 14 15 16
(week-old)
Supplementary Fig. S2
TNF-α (17 kDa)
IL-1β (35 kDa)
Repeat 1
Repeat 1
Repeat 2
20 -
Repeat 2
48 -
17 35 11 1
25 20 17 -
β-actin (45 kDa)
IL-6 (22–28 kDa)
Repeat 1
Repeat 1
Repeat 2
Repeat 2
63 -
48 -
48 -
25 20 -
35 -
17 25 1
Supplementary Fig. S3
**
3.5
Skp1 (Fold change)
gene expression (Fold change)
NC-miR mimic
2.5
miR-582-5p mimic
1.5
**
0.5
1.5
0.5
Myd88 Irak4
Skp1 (Fold change)
Traf6
Tak1
Tab1
Skp1
1.5
Tab2
Control
miR-582-5p miR-582-5p
mimic
inhibitor
DAPI
NC-miR
mimic
0.5
miR-582-5p
mimic
Control
LPS
β-actin (45 kDa)
SKP1 (19 kDa)
Repeat 1
Repeat 2
Repeat 1
20 -
63 48 -
17 -
35 -
25 -
NC miR NC miR
Repeat 2
NC miR NC miR
Supplementary Fig. S4
CUL1
RBX1
β-actin
NC-miR miR-582-5p
mimic
mimic
1.5
ns
0.5
NC-miR miR-582-5p
mimic
mimic
βTrCP (60 kDa)
Repeat 1
ns
1.5
Fold change (RBX1)
βTrCP
Fold change (CUL1)
Fold change (βTrCP)
0.5
NC-miR miR-582-5p
mimic
mimic
1.5
ns
0.5
NC-miR miR-582-5p
mimic
mimic
CUL1 (85 kDa)
Repeat 2
Repeat 1 Repeat 2
75 -
135 -
63 -
100 -
48 -
75 63 -
35 48 25 20 -
35 NC miR NC miR
NC miR NC miR
β-actin (45 kDa)
RBX1 (17 kDa)
Repeat 1
Repeat 1
Repeat 2
Repeat 2
63 -
25 48 -
20 17 -
35 NC miR NC miR
NC miR NC miR
Supplementary Fig. S5
IκBα (39 kDa)
SKP1 (19 kDa)
Repeat 1
Repeat 1
Repeat 2
35 25 20 -
63 48 -
17 -
35 -
11 1
Repeat 2
p65 (65 kDa)
p-p65 (65 kDa)
Repeat 1
Repeat 1
Repeat 2
Repeat 2
75 -
100 75 -
63 48 35 -
63 48 -
25 1
β-actin (45 kDa)
Repeat 1
Repeat 2
48 35 25 -
p65 (65 kDa) in nucleus
Repeat 1
Histone H1 (32–33 kDa)
Repeat 1
Repeat 2
Repeat 2
48 -
75 63 -
35 25 -
48 -
35 1
Supplementary Fig. S6
IκBα (39 kDa)
SKP1 (19 kDa)
Repeat 1
Repeat 1
Repeat 2
Repeat 2
25 20 63 48 -
17 11 1
35 -
p-p65 (65 kDa)
Repeat 1
25 -
Repeat 2
75 -
p65 (65 kDa)
63 -
Repeat 1
48 -
Repeat 2
35 1
β-actin (45 kDa)
Repeat 1
75 -
63 48 -
Repeat 2
35 -
63 48 -
35 25 1
p65 (65 kDa) in nucleus
LaminB1 (66 kDa)
Repeat 2
Repeat 1
75 -
75 63 -
63 48 -
48 -
63 48 1
100 75 63 48 35 -
75 -
35 -
35 -
Repeat 2
Repeat 1
Supplementary Fig. S7
p65
Merge
(DAPI, p65)
LPS
NC-miR mimic
miR-582-5p mimic
Supplementary Figure Legends
Supplementary Fig. S1. To study HFD-induced obesity, male C57BL/6J mice were fed with ND
or HFD for 8 weeks until 16 weeks of age. (A) Representative photo of mice after 8 weeks of ND
or HFD feeding. (B, C) Measurements of food intake (B) and body weight (C).
Supplementary Fig. S2. RAW264.7 cells were transfected with the control microRNA mimic
(lane 2) or the miR-582-5p mimic (lane 3) for 24 h, followed by incubation with (lane 2 and 3) or
without (lane 1) LPS for 4 h. The molecular weights of TNF-α, IL-1β, IL-6, and β-actin are 17
kDa, 35 kDa, 22–28 kDa, and 45 kDa, respectively. Molecular weight markers (in thousands) are
shown on the left-hand side of each blot. Arrowheads represent the position of each
immunoreactive band.
Supplementary Fig. S3. (A) RAW264.7 cells were transfected with the miR-582-5p mimic (20
nM) or its corresponding negative control microRNA mimic (NC-miR mimic, 20 nM) for 24 h.
Then, mRNA was isolated and subjected to quantitative real-time PCR analysis. Bar graphs
represent the mean ± SD (n = 3 for each group) relative to the NC-miR mimic transfected group.
Gapdh was used as the internal reference gene. **p < 0.01 represent significant differences
between the indicated bars (Student’s t-test). Myd88: myeloid differentiation primary response
gene 88; Irak4: interleukin-1 receptor-associated kinase 4; Traf6: TNF receptor-associated factor
6; Tak1: mitogen-activated protein kinase kinase kinase 7; Tab1: TGF-beta activated kinase
1/MAP3K7 binding protein 1; Tab2: TGF-beta activated kinase 1/MAP3K7 binding protein 2;
Skp1: S-phase kinase-associated protein 1. (B) RAW264.7 cells were transfected with the miR582-5p mimic (20 nM) or the miR-582-5p inhibitor (20 nM) for 24 h. Then, mRNA was isolated
and subjected to quantitative real-time PCR analysis. Bar graphs represent the mean ± SD (n = 3
for each group). Gapdh was used as the internal reference gene. *p < 0.05, **p < 0.01, represent
significant differences between the indicated bars (Tukey-Kramer’s HSD-test). (C) RAW264.7
cells were incubated with (LPS) or without (Control) LPS for 2 h. Then, mRNA was isolated, and
Skp1 mRNA expression was measured using quantitative real-time PCR analysis. We repeated
this experiment thrice. Gapdh was used as the internal reference gene. Bar graphs represent the
mean ± SD (n = 3 for each group). *p < 0.05 represent significant differences between the
indicated bars (Student’s t-test). (D) RAW264.7 cells were transfected with the miR-582-5p
mimic (20 nM) or the negative control microRNA mimic (NC-miR mimic, 20 nM) for 24 h. The
nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. (E) Two other data sets (Repeat
1 and 2) of Fig. 3D are shown. The molecular weights of SKP1 and β-actin are 19 kDa and 45
kDa, respectively. NC and miR represent the data from cells transfected with the negative control
microRNA mimic and miR-582-5p mimic, respectively. Molecular weight markers (in thousands)
are shown on the left-hand side of each blot. Arrowheads represent the position of each
immunoreactive band.
Supplementary Fig. S4. (A) RAW264.7 cells were transfected with either the miR-582-5p mimic
(20 nM) or its corresponding negative control microRNA mimic (NC-miR mimic, 20 nM) for 24
h. The protein levels of βTrCP, CUL1, and RBX1 were examined using western blotting. A set of
representative blots from three independent experiments are shown. β-actin was used as the
loading control. Bar graphs represent the mean ± SD (n = 3 for each group). ns, not significant
(Student’s t-test). (B) We repeated the above experiment thrice. The other two data sets are shown
as repeat 1 and 2 in (B). Molecular weight markers (in thousands) are shown on the left-hand side
of each blot. The molecular weights of βTrCP, CUL1, RBX1, and β-actin are 60 kDa, 85 kDa, 17
kDa, and 45 kDa, respectively. Arrowheads represent the position of each immunoreactive band.
NC and miR represent the data from cells transfected with the negative control microRNA mimic
and miR-582-5p mimic, respectively. βTrCP: beta transducin repeat-containing protein; CUL1:
cullin 1; RBX1: ring-box 1.
Supplementary Fig. S5. (A, B) RAW264.7 cells were transfected with si-control (lane 2) or siSkp1 (lane 3) for 24 h, followed by incubation with (lane 2, 3) or without (lane 1) LPS for 90 min;
cell lysates (A) or nuclear fraction (B) were analyzed. We repeated this experiment thrice; one set
of data is shown in Fig. 4B and C, and the other two other original images (Repeat 1 and 2) are
shown (A, B). The molecular weights of SKP1, IκBα, p65 (p-p65), β-actin, and histone H1 are 19
kDa, 39 kDa, 65 kDa, 45 kDa, and 32–33 kDa, respectively. Molecular weight markers (in
thousands) are shown on the left-hand side of each blot. Arrowheads represent the position of
each immunoreactive band.
Supplementary Fig. S6. (A, B) RAW264.7 cells were transfected with the control microRNA
mimic (lane 2) or the miR-582-5p mimic (lane 3) for 24 h, followed by the incubation with (lane
2, 3) or without (lane 1) LPS for 90 min; cell lysates (A) or nuclear fraction (B) were analyzed.
We repeated these experiments thrice; one set of data is shown in Fig. 4D and E, and the other
two original images (Repeat 1 and 2) are shown (A, B). The molecular weights of SKP1, IκBα,
p65 (p-p65), β-actin, and lamin B1 are 19 kDa, 39 kDa, 65 kDa, 45 kDa, and 66 kDa, respectively.
Molecular weight markers (in thousands) are shown on the left-hand side of each blot.
Arrowheads represent the position of each immunoreactive band.
Supplementary Fig. S7. RAW264.7 cells were transfected with the miR-582-5p mimic (20 nM)
or negative control microRNA mimic (NC-miR mimic, 20 nM) for 24 h and then incubated with
or without LPS for 90 min. The cells were analyzed through immunofluorescence staining using
an anti-p65 antibody. Two sets of images of p65 immunofluorescence staining (red) are shown.
The nuclei were counterstained with DAPI (blue). Scale bar = 20 µm.
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