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Expanded View Figures
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Figure EV1. PDGF induces FoxO3phosphorylation and nuclear exclusion of FoxO3.
A Densitometry quantitation ratio of p-FoxO3(Thr32) (left panel) and p-FoxO3(Ser253) (right panel) in serum-starved (48 h) N-HLF (n=3) that were stimulated
without/with PDGF-BB as indicated. Quantification is represented as a fold change to control (time corresponded non-stimulated cells). Data were analyzed using
repeated-measures ANOVA, **P<0.01 versus control 15 min,
§
P<0.05,
§§
P<0.01,
§§§
P<0.001 versus control 30 min.
B ICC of FoxO3in N-HLF that was serum-starved for 48 h, and stimulated with PDGF-BB (60 ng/ml) as indicated. Control image panel represents cells that were left
non-stimulated for 6h. TO-PRO3(blue) was used to label nuclei. FoxO3and TO-PRO3images were overlaid to visualize nuclear and cytoplasmic localization of FoxO3.
Images are representative of n=3. Scale bar = 50 lm.
ª2017 The Authors EMBO Molecular Medicine
Hamza M Al-Tamari et al FoxO3in lung fibrosis EMBO Molecular Medicine
EV1
A
C
B
Figure EV2. Different N-HLFs respond in similar
manner to various growth factors.
A–C Western blots of p-FoxO3(Thr32), FoxO3, and
GAPDH in serum-starved (48 h) N-HLF (n=3)
that was stimulated with 5% FCS (A), PDGF-BB
(B), or IGF-1(C) as indicated. Densitometry
quantified data of p-FoxO3(Thr32) to FoxO3
expression ratios, represented as a fold change
to non-stimulated cells.
▸
Figure EV3. Foxo3knockout (global- and fibroblast-specific) influences immune cell composition in bleomycin-instilled mice lungs.
A–D Immunofluorescence staining was performed on WT, Foxo3
/
and Foxo3
f.b
/
mice lung sections (saline- and bleomycin-instilled) using CD68,CD45, and CD3
antibodies. Representative pictographs depicting CD68 (A), CD45 (B), and CD3(C) staining in green from mice (n=3) in each group. DAPI was used as a nuclear
stain. Scale bar = 50 lm. Fluorescence intensities of CD3-stained sections (n=5/6per group) were quantified using ImageJ software and normalized to DAPI
intensity. Data are expressed as mean SEM and were analyzed using repeated-measures one-way ANOVA, ***P<0.001 versus WT saline group and
§§
P<0.01
versus WT bleomycin group. m1,m2, and m3represent three different mice evaluated in each group.
EMBO Molecular Medicine ª2017 The Authors
EMBO Molecular Medicine FoxO3in lung fibrosis Hamza M Al-Tamari et al
EV2
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C
D
B
Figure EV3.
ª2017 The Authors EMBO Molecular Medicine
Hamza M Al-Tamari et al FoxO3in lung fibrosis EMBO Molecular Medicine
EV3
Figure EV4. UCN-01 inhibits FoxO3nuclear exclusion of FCS-, IGF-1- or PDGF-BB- stimulated IPF-HLF.
Serum-starved (48 h) IPF-HLF were stimulated with 5% FCS or PDGF-BB (60 ng/ml) or IGF-1 (200 ng/ml) and treated with 50 nM UCN-01 or vehicle control (DMSO). After
6 h of treatment, ICC assessed FoxO3 cellular localization. Control panel represents cells that were treated for 6 h with 50 nM UCN-01 or vehicle control (DMSO). TO-PRO3
(blue) was used to label nuclei. FoxO3 and TO-PRO3 images were overlaid to visualize nuclear and cytoplasmic localization of FoxO3. Scale bar =50 lm. Images are
representative of n=3.
A B
Figure EV5. Anti-proliferative effects of UCN-01 are mediated via FoxO3.
A, B N-HLF was transfected with empty vector (EV) or AKT mutant (AKT mut) plasmid. 6h after transfection, cells were serum-starved for 36 h and then stimulated with
5% FCS in the presence or absence of UCN-01. From the above-treated samples, after 24 h, Western blots [p-FoxO3(Thr32), FoxO3, AKT, AKT mut, GAPDH] and cell
proliferation measurements (BrdU incorporation) were performed. Data represent percentage of control, EV non-stimulated cells (n=3). Data are expressed as
mean SEM and were analyzed using one-way ANOVA, *P<0.05,**P<0.01, and ***P<0.001 versus 5% FCS-EV, and
§§§
P<0.001 versus UCN-01-EV.
EMBO Molecular Medicine ª2017 The Authors
EMBO Molecular Medicine FoxO3in lung fibrosis Hamza M Al-Tamari et al
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